Compositions and methods for the suppression of target polynucleotides from lepidoptera

ABSTRACT

Methods and compositions are provided which employ a silencing element that, when ingested by a pest, such as a pest from the Lepidoptera order, they are capable of decreasing the expression of a target sequence in the pest. Further provided are silencing elements which when ingested by the pest decrease the level of the target polypeptide and thereby control the pest. In specific embodiment, the pest is  Spodoptera frugiperda . Plants, plant part, bacteria and other host cells comprising the silencing elements or an active variant or fragment thereof of the invention are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, U.S. Provisional Application No.61/021,699, filed Jan. 17, 2008, and U.S. Provisional Application No.61/021,676, filed Jan. 17, 2008; and is a continuation of U.S. NonProvisional application Ser. No. 12/351,267 filed Jan. 9, 2009, nowgranted as U.S. Pat. No. 8,847,013, which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to methods of molecular biologyand gene silencing to control pests.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 366590seqlist.txt, a creation date of Jan. 9, 2009, and a sizeof 102 Kb. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

Insect pests are a serious problem in agriculture. They destroy millionsof acres of staple crops such as corn, soybeans, peas, and cotton.Yearly, these pests cause over $100 billion dollars in crop damage inthe U.S. alone. In an ongoing seasonal battle, farmers must applybillions of gallons of synthetic pesticides to combat these pests. Othermethods employed in the past delivered insecticidal activity bymicroorganisms or genes derived from microorganisms expressed intransgenic plants. For example, certain species of microorganisms of thegenus Bacillus are known to possess pesticidal activity against a broadrange of insect pests including Lepidoptera, Diptera, Coleoptera,Hemiptera, and others. In fact, microbial pesticides, particularly thoseobtained from Bacillus strains, have played an important role inagriculture as alternatives to chemical pest control. Agriculturalscientists have developed crop plants with enhanced insect resistance bygenetically engineering crop plants to produce insecticidal proteinsfrom Bacillus. For example, corn and cotton plants geneticallyengineered to produce Cry toxins (see, e.g., Aronson (2002) Cell Mol.Life Sci. 59(3):417-425; Schnepf et al. (1998) Microbiol. Mol. Biol.Rev. 62(3):775-806) are now widely used in American agriculture and haveprovided the farmer with an alternative to traditional insect-controlmethods. However, these Bt insecticidal proteins only protect plantsfrom a relatively narrow range of pests. Moreover, these modes ofinsecticidal activity provided varying levels of specificity and, insome cases, caused significant environmental consequences. Thus, thereis an immediate need for alternative methods to control pests.

BRIEF SUMMARY OF THE INVENTION

Methods and compositions are provided which employ a silencing elementthat, when ingested by a pest, such as a pest from the Lepidopteraorder, is capable of decreasing the expression of a target sequence inthe pest. In specific embodiments, the decrease in expression of thetarget sequence controls the pest and thereby the methods andcompositions are capable of limiting damage to a plant. The presentinvention provides various target polynucleotides encoding polypeptidesfrom specific families as disclosed elsewhere herein and various targetpolynucleotides set forth in SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 or active variants or fragments thereof, wherein adecrease in expression of one or more the sequences in the target pestcontrols the pest (i.e., has insecticidal activity). Further providedare silencing elements, which when ingested by the pest, decrease thelevel of expression of one or more of the target polynucleotides. Inspecific embodiments, the silencing element comprises at least 15, 20,or 22 consecutive nucleotides of any one of SEQ ID NO:1-50. In specificembodiments, the pest that is controlled is Spodoptera frugiperda.Plants, plant parts, plant cells, bacteria and other host cellscomprising the silencing elements or an active variant or fragmentthereof are also provided.

In another embodiment, a method for controlling a pest, such as a pestfrom the Lepidoptera order, is provided. The method comprises feeding toa pest a composition comprising a silencing element, wherein thesilencing element, when ingested by the pest, reduces the level of atarget sequence in the pest and thereby controls the pest. Furtherprovided are methods to protect a plant from a pest. Such methodscomprise introducing into the plant or plant part a silencing element ofthe invention. When the plant expressing the silencing element isingested by the pest, the level of the target sequence is decreased andthe pest is controlled.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

I. Overview

Methods and compositions are provided which employ a silencing elementthat, when ingested by a pest, such as a pest from the Lepidopteraorder, is capable of decreasing the expression of a target sequence inthe pest. In specific embodiments, the decrease in expression of thetarget sequence controls the pest and thereby the methods andcompositions are capable of limiting damage to a plant or plant part.The present invention provides target polynucleotides which encodepolypeptides from a variety of protein classes including, for example, ajuvenile hormone polypeptide, a vacuolar polypeptide, a cadherinpolypeptide, a cuticle polypeptide, a translation initiation factor, aSARI polypeptide, an elongation factor, a phosphooligosaccharide, amyosin polypeptide, a potassium channel amino acid transporter, apotassium inwardly rectifier polypeptide, an amino acid transporter, atubulin polypeptide, a ubiquitin polypeptide, and small nuclearribonucleoprotein. In other embodiments the target polynucleotides areset forth in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 or active variants and fragments thereof. Silencing elements designedin view of these target polynucleotides are provided which, wheningested by the pest, decrease the expression of one or more of thetarget sequences and thereby controls the pest (i.e., has insecticidalactivity). See, for example, SEQ ID NOS:51-465.

As used herein, by “controlling a pest” or “controls a pest” is intendedany affect on a pest that results in limiting the damage that the pestcauses. Controlling a pest includes, but is not limited to, killing thepest, inhibiting development of the pest, altering fertility or growthof the pest in such a manner that the pest provides less damage to theplant, decreasing the number of offspring produced, producing less fitpests, producing pests more susceptible to predator attack, or deterringthe pests from eating the plant.

By “disease resistance” is intended that the plants avoid the diseasesymptoms that are the outcome of plant-pathogen interactions. That is,pathogens are prevented from causing plant diseases and the associateddisease symptoms, or alternatively, the disease symptoms caused by thepathogen is minimized or lessened.

Reducing the level of expression of the target polynucleotide or thepolypeptide encoded thereby, in the pest results in the suppression,control, and/or killing the invading pathogenic organism. Reducing thelevel of expression of the target sequence of the pest will reduce thedisease symptoms resulting from pathogen challenge by at least about 2%to at least about 6%, at least about 5% to about 50%, at least about 10%to about 60%, at least about 30% to about 70%, at least about 40% toabout 80%, or at least about 50% to about 90% or greater. Hence, themethods of the invention can be utilized to protect plants from disease,particularly those diseases that are caused by pests from theLepidoptera order.

Assays that measure the control of a pest are commonly known in the art,as are methods to quantitate disease resistance in plants followingpathogen infection. See, for example, U.S. Pat. No. 5,614,395, hereinincorporated by reference. Such techniques include, measuring over time,the average lesion diameter, the pathogen biomass, and the overallpercentage of decayed plant tissues. See, for example, Thomma et al.(1998) Plant Biology 95:15107-15111, herein incorporated by reference.See, also the examples below.

The invention is drawn to compositions and methods for protecting plantsfrom a plant pest, such as pests from the Lepidoptera order or inducingresistance in a plant to a plant pest, such as pests from theLepidoptera order. Caterpillars and related forms of lepidopteraninsects comprise an important group of plant-feeding agricultural pests,especially during the larvae stage of growth. Feeding methods ofLepidoptera larvae typically include chewing plants or plant parts. Asused herein, the term “Lepidoptera” is used to refer to any member ofthe Lepidoptera order. In particular embodiments, compositions andmethods of the invention control Lepidoptera larvae (i.e. caterpillars).Accordingly, the compositions and methods are also useful in protectingplants against any Lepidoptera including, for example, Pieris rapae,Pectinophora gossypiella, Synanthedon exitiosa, Melittia cucurbitae,Cydia pomonella, Grapholita molesta, Ostrinia nubilalis, Plodiainterpunctella, Galleria mellonella, Manduca sexta, Manducaquinquemaculata, Lymantria dispar, Euproctis chrysorrhoea, Trichoplusiani, Mamestra brassicae, Agrotis ipsilon, Plutella xylostella, Anticarsiagemmatalis, Psuedoplusia includens, Epinotia aporema, Helicoverpa zea,Heliothis virescens, Heliothis armigera, Spodoptera exigua, Scirpophagaincertulus, Sesamia spp., Buseola fusca, Cnaphalocrocis medinalis, Chilosuppressalis, or Spodoptera littoralis. In particular embodiments,methods control Spodoptera frugiperda.

II. Target Sequences

As used herein, a “target sequence” or “target polynucleotide” comprisesany sequence in the pest that one desires to reduce the level ofexpression. In specific embodiments, decreasing the level of the targetsequence in the pest controls the pest. For instance, the targetsequence can be essential for growth and development. While the targetsequence can be expressed in any tissue of the pest, in specificembodiments of the invention, the sequences targeted for suppression inthe pest are expressed in cells of the gut tissue of the pest, cells inthe midgut of the pest, and cells lining the gut lumen or the midgut.Such target sequences can be involved in gut cell metabolism, growth ordifferentiation.

In one embodiment of the invention the target sequence comprises apolypeptide belonging to one or more classes of enzymes such as ajuvenile hormone polypeptide, a vacuolar polypeptide, a cadherinpolypeptide, a cuticle polypeptide, a translation initiation factor, aSARI polypeptide, an elongation factor, a phosphooligosaccharide, amyosin polypeptide, a potassium channel amino acid transporter, apotassium inwardly rectifier, an amino acid transporter, a tubulinpolypeptide, a ubiquitin polypeptide, and a small nuclearribonucleoprotein. Non-limiting examples of target sequences of theinvention include a polynucleotide set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or 50. As exemplified elsewhere herein,decreasing the level of expression of these target sequence or membersof the recited enzyme classes in Lepidoptera controls the pest.

III. Silencing Elements

By “silencing element” is intended a polynucleotide which when ingestedby a pest, is capable of reducing or eliminating the level or expressionof a target polynucleotide or the polypeptide encoded thereby. Thesilencing element employed can reduce or eliminate the expression levelof the target sequence by influencing the level of the target RNAtranscript or, alternatively, by influencing translation and therebyaffecting the level of the encoded polypeptide. Methods to assay forfunctional silencing elements that are capable of reducing oreliminating the level of a sequence of interest are disclosed elsewhereherein. A single polynucleotide employed in the methods of the inventioncan comprises one or more silencing elements to the same or differenttarget polynucleotides.

In specific embodiments, the target sequence is not a plant endogenousgene. In other embodiments, while the silencing element controls pests,preferably the silencing element has no effect on the normal plant orplant part.

As discussed in further detail below, silencing elements can include,but are not limited to, a sense suppression element, an antisensesuppression element, a double stranded RNA, a miRNA, or a hairpinsuppression element. Non-limiting examples of silencing elements thatcan be employed to decrease expression of these target Lepidopterasequences comprise fragments and variants of the sense or antisensesequence or consists of the sense or antisense sequence of the sequenceset forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102,105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144,147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186,189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228,231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270,273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312,315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354,357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396,399, 402, 405, 408, 411, 415, 418, 421, 424, 427, 430, 433, 436, 439,442, 457, 460, and/or 463 or a biologically active variant or fragmentthereof. In specific embodiments, the silencing element comprises orconsists of at least one of the sequences set forth in any one of SEQ IDNOS: 51-465. In further embodiments, the silencing elements can compriseat least one thymine residue at the 3′ end. This can aid instabilization. Thus, the silencing elements can have at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more thymine residues at the 3′ end.

In further embodiments, the silencing element comprises SEQ ID NO: 52and 53; 55 and 56; 58 and 59; 61 and 62; 64 and 65; 67 and 68; 70 and71; 73 and 74; 76 and 77; 79 and 80; 82 and 83; 85 and 86; 88 and 89; 91and 92; 94 and 95; 97 and 98; 100 and 101; 103 and 104; 106 and 107; 109and 110; 112 and 113; 115 and 116; 118 and 119; 121 and 122; 124 and125; 127 and 128; 130 and 131; 133 and 134; 136 and 137; 139 and 140;142 and 143; 145 and 146; 148 and 149; 151 and 152; 154 and 155; 157 and158; 160 and 161; 163 and 164; 166 and 167; 169 and 170; 172 and 173;175 and 176; 178 and 179; 181 and 182; 184 and 185; 187 and 188; 190 and191; 193 and 194; 196 and 197; 199 and 200; 202 and 203; 205 and 206;208 and 209; 211 and 212; 214 and 215; 217 and 218; 220 and 221; 223 and224; 226 and 227; 229 and 230; 232 and 233; 235 and 236; 238 and 239;241 and 242; 244 and 245; 247 and 248; 250 and 251; 253 and 254; 256 and257; 259 and 260; 262 and 263; 265 and 266; 268 and 269; 271 and 272;274 and 275; 277 and 278; 280 and 281; 283 and 284; 286 and 287; 289 and290; 292 and 293; 295 and 296; 298 and 299; 301 and 302; 304 and 305;307 and 308; 310 and 311; 313 and 314; 316 and 317; 139 and 320; 322 and323; 325 and 326; 328 and 329; 331 and 332; 334 and 335; 337 and 338;340 and 341; 343 and 344; 346 and 347; 349 and 350; 352 and 353; 355 and356; 358 and 359; 361 and 362; 364 and 365; 367 and 368; 370 and 371;373 and 374; 376 and 377; 379 and 380; 382 and 383; 385 and 386; 388 and389; 391 and 392; 394 and 395; 397 and 398; 400 and 401; 403 and 404;406 and 407; 409 and 410; 412 and 413; 416 and 417; 419 and 420; 422 and423; 425 and 426; 428 and 429; 431 and 432; 434 and 435; 437 and 438;440 and 441; 443 and 444; 458 and 459; 461 and 462; and/or 464 and 465.

By “reduces” or “reducing” the expression level of a polynucleotide or apolypeptide encoded thereby is intended to mean, the polynucleotide orpolypeptide level of the target sequence is statistically lower than thepolynucleotide level or polypeptide level of the same target sequence inan appropriate control pest which is not exposed to (i.e., has notingested) the silencing element. In particular embodiments of theinvention, reducing the polynucleotide level and/or the polypeptidelevel of the target sequence in a pest according to the inventionresults in less than 95%, less than 90%, less than 80%, less than 70%,less than 60%, less than 50%, less than 40%, less than 30%, less than20%, less than 10%, or less than 5% of the polynucleotide level, or thelevel of the polypeptide encoded thereby, of the same target sequence inan appropriate control pest. Methods to assay for the level of the RNAtranscript, the level of the encoded polypeptide, or the activity of thepolynucleotide or polypeptide are discussed elsewhere herein.

i. Sense Suppression Elements

As used herein, a “sense suppression element” comprises a polynucleotidedesigned to express an RNA molecule corresponding to at least a part ofa target messenger RNA in the “sense” orientation. Expression of the RNAmolecule comprising the sense suppression element reduces or eliminatesthe level of the target polynucleotide or the polypeptide encodedthereby. The polynucleotide comprising the sense suppression element maycorrespond to all or part of the sequence of the target polynucleotide,all or part of the 5′ and/or 3′ untranslated region of the targetpolynucleotide, all or part of the coding sequence of the targetpolynucleotide, or all or part of both the coding sequence and theuntranslated regions of the target polynucleotide.

Typically, a sense suppression element has substantial sequence identityto the target polynucleotide, typically greater than about 65% sequenceidentity, greater than about 85% sequence identity, about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. See, U.S. Pat.Nos. 5,283,184 and 5,034,323; herein incorporated by reference. Thesense suppression element can be any length so long as it allows for thesuppression of the targeted sequence. The sense suppression element canbe, for example, 15, 20, 22, 25, 30, 50, 100, 150, 200, 250, 300, 350,400, 450, 500, 600, 700, 900 or longer.

ii. Antisense Suppression Elements

As used herein, an “antisense suppression element” comprises apolynucleotide which is designed to express an RNA moleculecomplementary to all or part of a target messenger RNA. Expression ofthe antisense RNA suppression element reduces or eliminates the level ofthe target polynucleotide. The polynucleotide for use in antisensesuppression may correspond to all or part of the complement of thesequence encoding the target polynucleotide, all or part of thecomplement of the 5′ and/or 3′ untranslated region of the targetpolynucleotide, all or part of the complement of the coding sequence ofthe target polynucleotide, or all or part of the complement of both thecoding sequence and the untranslated regions of the targetpolynucleotide. In addition, the antisense suppression element may befully complementary (i.e., 100% identical to the complement of thetarget sequence) or partially complementary (i.e., less than 100%identical to the complement of the target sequence) to the targetpolynucleotide. In specific embodiments, the antisense suppressionelement comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence complementarity to the target polynucleotide.Antisense suppression may be used to inhibit the expression of multipleproteins in the same plant. See, for example, U.S. Pat. No. 5,942,657.Furthermore, the antisense suppression element can be complementary to aportion of the target polynucleotide. Generally, sequences of at least15, 20, 22, 25, 50, 100, 200, 300, 400, 450 nucleotides or greater maybe used. Methods for using antisense suppression to inhibit theexpression of endogenous genes in plants are described, for example, inLiu et al (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos.5,759,829 and 5,942,657, each of which is herein incorporated byreference.

iii. Double Stranded RNA Suppression Element

A “double stranded RNA silencing element” or “dsRNA” comprises at leastone transcript that is capable of forming a dsRNA either before or afteringestion by a pest. Thus, a “dsRNA silencing element” includes a dsRNA,a transcript or polyribonucleotide capable of forming a dsRNA or morethan one transcript or polyribonucleotide capable of forming a dsRNA.“Double stranded RNA” or “dsRNA” refers to a polyribonucleotidestructure formed either by a single self-complementary RNA molecule or apolyribonucleotide structure formed by the expression of least twodistinct RNA strands. The dsRNA molecule(s) employed in the methods andcompositions of the invention mediate the reduction of expression of atarget sequence, for example, by mediating RNA interference “RNAi” orgene silencing in a sequence-specific manner. In the context of thepresent invention, the dsRNA is capable of reducing or eliminating thelevel or expression of a target polynucleotide or the polypeptideencoded thereby in a pest.

The dsRNA can reduce or eliminate the expression level of the targetsequence by influencing the level of the target RNA transcript, byinfluencing translation and thereby affecting the level of the encodedpolypeptide, or by influencing expression at the pre-transcriptionallevel (i.e., via the modulation of chromatin structure, methylationpattern, etc., to alter gene expression). See, for example, Verdel etal. (2004) Science 303:672-676; Pal-Bhadra et al. (2004) Science303:669-672; Allshire (2002) Science 297:1818-1819; Volpe et al. (2002)Science 297:1833-1837; Jenuwein (2002) Science 297:2215-2218; and Hallet al. (2002) Science 297:2232-2237. Methods to assay for functionaliRNA that are capable of reducing or eliminating the level of a sequenceof interest are disclosed elsewhere herein. Accordingly, as used herein,the term “dsRNA” is meant to encompass other terms used to describenucleic acid molecules that are capable of mediating RNA interference orgene silencing, including, for example, short-interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), hairpin RNA, shorthairpin RNA (shRNA), post-transcriptional gene silencing RNA (ptgsRNA),and others.

In specific embodiments, at least one strand of the duplex ordouble-stranded region of the dsRNA shares sufficient sequence identityor sequence complementarity to the target polynucleotide to allow forthe dsRNA to reduce the level of expression of the target sequence. Asused herein, the strand that is complementary to the targetpolynucleotide is the “antisense strand” and the strand homologous tothe target polynucleotide is the “sense strand.”

In one embodiment, the dsRNA comprises a hairpin RNA. A hairpin RNAcomprises an RNA molecule that is capable of folding back onto itself toform a double stranded structure. Multiple structures can be employed ashairpin elements. In specific embodiments, the dsRNA suppression elementcomprises a hairpin element which comprises in the following order, afirst segment, a second segment, and a third segment, where the firstand the third segment share sufficient complementarity to allow thetranscribed RNA to form a double-stranded stem-loop structure.

The “second segment” of the hairpin comprises a “loop” or a “loopregion.” These terms are used synonymously herein and are to beconstrued broadly to comprise any nucleotide sequence that confersenough flexibility to allow self-pairing to occur between complementaryregions of a polynucleotide (i.e., segments 1 and 3 which form the stemof the hairpin). For example, in some embodiments, the loop region maybe substantially single stranded and act as a spacer between theself-complementary regions of the hairpin stem-loop. In someembodiments, the loop region can comprise a random or nonsensenucleotide sequence and thus not share sequence identity to a targetpolynucleotide. In other embodiments, the loop region comprises a senseor an antisense RNA sequence or fragment thereof that shares identity toa target polynucleotide. See, for example, International PatentPublication No. WO 02/00904, herein incorporated by reference. Inspecific embodiments, the loop region can be optimized to be as short aspossible while still providing enough intramolecular flexibility toallow the formation of the base-paired stem region. Accordingly, theloop sequence is generally less than 1000, 900, 800, 700, 600, 500, 400,300, 200, 100, 50, 25, 20, 15, 10 nucleotides or less.

The “first” and the “third” segment of the hairpin RNA molecule comprisethe base-paired stem of the hairpin structure. The first and the thirdsegments are inverted repeats of one another and share sufficientcomplementarity to allow the formation of the base-paired stem region.In specific embodiments, the first and the third segments are fullycomplementary to one another. Alternatively, the first and the thirdsegment may be partially complementary to each other so long as they arecapable of hybridizing to one another to form a base-paired stem region.The amount of complementarity between the first and the third segmentcan be calculated as a percentage of the entire segment. Thus, the firstand the third segment of the hairpin RNA generally share at least 50%,60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, upto and including 100% complementarity.

The first and the third segment are at least about 1000, 500, 400, 300,200, 100, 50, 40, 30, 25, 22, 20, 15 or 10 nucleotides in length. Inspecific embodiments, the length of the first and/or the third segmentis about 10-100 nucleotides, about 10 to about 75 nucleotides, about 10to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 toabout 35 nucleotides, about 10 to about 30 nucleotides, about 10 toabout 25 nucleotides, about 10 to about 20 nucleotides. In otherembodiments, the length of the first and/or the third segment comprisesat least 10-20 nucleotides, 20-35 nucleotides, 30-45 nucleotides, 40-50nucleotides, 50-100 nucleotides, or 100-300 nucleotides. See, forexample, International Publication No. WO 0200904. In specificembodiments, the first and the third segment comprise at least 20nucleotides having at least 85% complementary to the first segment. Instill other embodiments, the first and the third segments which form thestem-loop structure of the hairpin comprises 3′ or 5′ overhang regionshaving unpaired nucleotide residues.

In specific embodiments, the sequences used in the first, the second,and/or the third segments comprise domains that are designed to havesufficient sequence identity to a target polynucleotide of interest andthereby have the ability to decrease the level of expression of thetarget polynucleotide. The specificity of the inhibitory RNA transcriptsis therefore generally conferred by these domains of the silencingelement. Thus, in some embodiments of the invention, the first, secondand/or third segment of the silencing element comprise a domain havingat least 10, at least 15, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 30, at least40, at least 50, at least 100, at least 200, at least 300, at least 500,at least 1000, or more than 1000 nucleotides that share sufficientsequence identity to the target polynucleotide to allow for a decreasein expression levels of the target polynucleotide when expressed in anappropriate cell. In other embodiments, the domain is between about 15to 50 nucleotides, about 20-35 nucleotides, about 25-50 nucleotides,about 20 to 75 nucleotides, about 40-90 nucleotides about 15-100nucleotides.

In specific embodiments, the domain of the first, the second, and/or thethird segment has 100% sequence identity to the target polynucleotide.In other embodiments, the domain of the first, the second and/or thethird segment having homology to the target polypeptide have at least50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or greater sequence identity to a region of the targetpolynucleotide. The sequence identity of the domains of the first, thesecond and/or the third segments to the target polynucleotide need onlybe sufficient to decrease expression of the target polynucleotide ofinterest. See, for example, Chuang and Meyerowitz (2000) Proc. Natl.Acad. Sci. USA 97:4985-4990; Stoutjesdijk et al. (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38;Pandolfini et al. BMC Biotechnology 3:7, and U.S. Patent Publication No.20030175965; each of which is herein incorporated by reference. Atransient assay for the efficiency of hpRNA constructs to silence geneexpression in vivo has been described by Panstruga et al. (2003) Mol.Biol. Rep. 30:135-140, herein incorporated by reference.

The amount of complementarity shared between the first, second, and/orthird segment and the target polynucleotide or the amount ofcomplementarity shared between the first segment and the third segment(i.e., the stem of the hairpin structure) may vary depending on theorganism in which gene expression is to be controlled. Some organisms orcell types may require exact pairing or 100% identity, while otherorganisms or cell types may tolerate some mismatching. In some cells,for example, a single nucleotide mismatch in the targeting sequenceabrogates the ability to suppress gene expression. In these cells, thesuppression cassettes of the invention can be used to target thesuppression of mutant genes, for example, oncogenes whose transcriptscomprise point mutations and therefore they can be specifically targetedusing the methods and compositions of the invention without altering theexpression of the remaining wild-type allele.

Any region of the target polynucleotide can be used to design the domainof the silencing element that shares sufficient sequence identity toallow expression of the hairpin transcript to decrease the level of thetarget polynucleotide. For instance, the domain can be designed to sharesequence identity to the 5′ untranslated region of the targetpolynucleotide(s), the 3′ untranslated region of the targetpolynucleotide(s), exonic regions of the target polynucleotide(s),intronic regions of the target polynucleotide(s), and any combinationthereof. In specific embodiments a domain of the silencing elementshares sufficient homology to at least about 15, 20, 22, 25 or 30consecutive nucleotides from about nucleotides 1-50, 50-100, 100-150,150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 550-600,600-650, 650-700, 750-800, 850-900, 950-1000, 1000-1050, 1050-1100,1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700,1700-1800, 1800-1900, 1900-2000 of the target sequence. In someinstances to optimize the siRNA sequences employed in the hairpin, thesynthetic oligodeoxyribonucleotide/RNAse H method can be used todetermine sites on the target mRNA that are in a conformation that issusceptible to RNA silencing. See, for example, Vickers et al. (2003) J.Biol. Chem 278:7108-7118 and Yang et al. (2002) Proc. Natl. Acad. Sci.USA 99:9442-9447, herein incorporated by reference. These studiesindicate that there is a significant correlation between theRNase-H-sensitive sites and sites that promote efficient siRNA-directedmRNA degradation.

The hairpin silencing element may also be designed such that the sensesequence or the antisense sequence do not correspond to a targetpolynucleotide. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the target polynucleotide. Thus, it is the loop regionthat determines the specificity of the RNA interference. See, forexample, WO 02/00904, herein incorporated by reference.

In specific embodiments, the silencing element comprising the hairpincomprises sequences selected from the group consisting of SEQ ID NO: 52and 53; 55 and 56; 58 and 59; 61 and 62; 64 and 65; 67 and 68; 70 and71; 73 and 74; 76 and 77; 79 and 80; 82 and 83; 85 and 86; 88 and 89; 91and 92; 94 and 95; 97 and 98; 100 and 101; 103 and 104; 106 and 107; 109and 110; 112 and 113; 115 and 116; 118 and 119; 121 and 122; 124 and125; 127 and 128; 130 and 131; 133 and 134; 136 and 137; 139 and 140;142 and 143; 145 and 146; 148 and 149; 151 and 152; 154 and 155; 157 and158; 160 and 161; 163 and 164; 166 and 167; 169 and 170; 172 and 173;175 and 176; 178 and 179; 181 and 182; 184 and 185; 187 and 188; 190 and191; 193 and 194; 196 and 197; 199 and 200; 202 and 203; 205 and 206;208 and 209; 211 and 212; 214 and 215; 217 and 218; 220 and 221; 223 and224; 226 and 227; 229 and 230; 232 and 233; 235 and 236; 238 and 239;241 and 242; 244 and 245; 247 and 248; 250 and 251; 253 and 254; 256 and257; 259 and 260; 262 and 263; 265 and 266; 268 and 269; 271 and 272;274 and 275; 277 and 278; 280 and 281; 283 and 284; 286 and 287; 289 and290; 292 and 293; 295 and 296; 298 and 299; 301 and 302; 304 and 305;307 and 308; 310 and 311; 313 and 314; 316 and 317; 139 and 320; 322 and323; 325 and 326; 328 and 329; 331 and 332; 334 and 335; 337 and 338;340 and 341; 343 and 344; 346 and 347; 349 and 350; 352 and 353; 355 and356; 358 and 359; 361 and 362; 364 and 365; 367 and 368; 370 and 371;373 and 374; 376 and 377; 379 and 380; 382 and 383; 385 and 386; 388 and389; 391 and 392; 394 and 395; 397 and 398; 400 and 401; 403 and 404;406 and 407; 409 and 410; 412 and 413; 416 and 417; 419 and 420; 422 and423; 425 and 426; 428 and 429; 431 and 432; 434 and 435; 437 and 438;440 and 441; 443 and 444; 458 and 459; 461 and 462; and/or 464 and 465.

In addition, transcriptional gene silencing (TGS) may be accomplishedthrough use of a hairpin suppression element where the inverted repeatof the hairpin shares sequence identity with the promoter region of atarget polynucleotide to be silenced. See, for example, Aufsatz et al.(2002) PNAS 99 (Suppl. 4):16499-16506 and Mette et al. (2000) EMBO J19(19):5194-5201.

In other embodiments, the dsRNA can comprise a small RNA (sRNA). sRNAscan comprise both micro RNA (miRNA) and short-interfering RNA (siRNA)(Meister and Tuschl (2004) Nature 431:343-349 and Bonetta et al. (2004)Nature Methods 1:79-86). miRNAs are regulatory agents comprising about19 ribonucleotides which are highly efficient at inhibiting theexpression of target polynucleotides. See, for example Javier et al.(2003) Nature 425: 257-263, herein incorporated by reference. For miRNAinterference, the silencing element can be designed to express a dsRNAmolecule that forms a hairpin structure containing a 19-nucleotidesequence that is complementary to the target polynucleotide of interest.The miRNA can be synthetically made, or transcribed as a longer RNAwhich is subsequently cleaved to produce the active miRNA. Specifically,the miRNA can comprise 19 nucleotides of the sequence having homology toa target polynucleotide in sense orientation and 19 nucleotides of acorresponding antisense sequence that is complementary to the sensesequence.

When expressing an miRNA, it is recognized that various forms of anmiRNA can be transcribed including, for example, the primary transcript(termed the “pri-miRNA”) which is processed through various nucleolyticsteps to a shorter precursor miRNA (termed the “pre-miRNA”); thepre-miRNA; or the final (mature) miRNA is present in a duplex, the twostrands being referred to as the miRNA (the strand that will eventuallybasepair with the target) and miRNA*. The pre-miRNA is a substrate for aform of dicer that removes the miRNA/miRNA* duplex from the precursor,after which, similarly to siRNAs, the duplex can be taken into the RISCcomplex. It has been demonstrated that miRNAs can be transgenicallyexpressed and be effective through expression of a precursor form,rather than the entire primary form (Parizotto et al. (2004) Genes &Development 18:2237-2242 and Guo et al. (2005) Plant Cell 17:1376-1386).

The methods and compositions of the invention employ silencing elementsthat when transcribed “form” a dsRNA molecule. Accordingly, theheterologous polynucleotide being expressed need not form the dsRNA byitself, but can interact with other sequences in the plant cell or inthe pest gut after ingestion to allow the formation of the dsRNA. Forexample, a chimeric polynucleotide that can selectively silence thetarget polynucleotide can be generated by expressing a chimericconstruct comprising the target sequence for a miRNA or siRNA to asequence corresponding to all or part of the gene or genes to besilenced. In this embodiment, the dsRNA is “formed” when the target forthe miRNA or siRNA interacts with the miRNA present in the cell. Theresulting dsRNA can then reduce the level of expression of the gene orgenes to be silenced. See, for example, U.S. Provisional Application No.60/691,613, filed Jun. 17, 2005, entitled “Methods and Compositions forGene Silencing, herein incorporated by reference. The construct can bedesigned to have a target for an endogenous miRNA or alternatively, atarget for a heterologous and/or synthetic miRNA can be employed in theconstruct. If a heterologous and/or synthetic miRNA is employed, it canbe introduced into the cell on the same nucleotide construct as thechimeric polynucleotide or on a separate construct. As discussedelsewhere herein, any method can be used to introduce the constructcomprising the heterologous miRNA.

IV. Variants and Fragments

By “fragment” is intended a portion of the polynucleotide or a portionof the amino acid sequence and hence protein encoded thereby. Fragmentsof a polynucleotide may encode protein fragments that retain thebiological activity of the native protein. Alternatively, fragments of apolynucleotide that are useful as a silencing element do not need toencode fragment proteins that retain biological activity. Thus,fragments of a nucleotide sequence may range from at least about 10,about 15, about 20 nucleotides, about 22 nucleotides, about 50nucleotides, about 75 nucleotides, about 100 nucleotides, 200nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600nucleotides, 700 nucleotides and up to the full-length polynucleotideemployed in the invention. Methods to assay for the activity of adesired silencing element or a suppressor enhancer element are describedelsewhere herein.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. For polynucleotides,conservative variants include those sequences that, because of thedegeneracy of the genetic code, encode the amino acid sequence of one ofthe polypeptides employed in the invention. Variant polynucleotides alsoinclude synthetically derived polynucleotide, such as those generated,for example, by using site-directed mutagenesis, but continue to retainthe desired activity. Generally, variants of a particular polynucleotideof the invention (i.e., a silencing element) will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular polynucleotide as determined by sequence alignment programsand parameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Percent sequence identity between any two polypeptidescan be calculated using sequence alignment programs and parametersdescribed elsewhere herein. Where any given pair of polynucleotidesemployed in the invention is evaluated by comparison of the percentsequence identity shared by the two polypeptides they encode, thepercent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins encompassed by the present invention are biologically active,that is they continue to possess the desired biological activity of thenative protein, as discussed elsewhere herein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.Biologically active variants of a native protein will have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the aminoacid sequence for the native protein as determined by sequence alignmentprograms and parameters described elsewhere herein. A biologicallyactive variant of a protein of the invention may differ from thatprotein by as few as 1-15 amino acid residues, as few as 1-10, such as6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

V. DNA Constructs

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

The polynucleotide encoding the silencing element or in specificembodiments employed in the methods and compositions of the inventioncan be provided in expression cassettes for expression in a plant ororganism of interest. It is recognized that multiple silencing elementsincluding multiple identical silencing elements, multiple silencingelements targeting different regions of the target sequence, or multiplesilencing elements from different target sequences can be used. In thisembodiment, it is recognized that each silencing element can becontained in a single or separate cassette, DNA construct, or vector. Asdiscussed, any means of providing the silencing element is contemplated.A plant or plant cell can be transformed with a single cassettecomprising DNA encoding one or more silencing elements or separatecassettes comprising each silencing element can be used to transform aplant or plant cell or host cell. Likewise, a plant transformed with onecomponent can be subsequently transformed with the second component. Oneor more silencing elements can also be brought together by sexualcrossing. That is, a first plant comprising one component is crossedwith a second plant comprising the second component. Progeny plants fromthe cross will comprise both components.

The expression cassette can include 5′ and 3′ regulatory sequencesoperably linked to the polynucleotide of the invention. “Operablylinked” is intended to mean a functional linkage between two or moreelements. For example, an operable linkage between a polynucleotide ofthe invention and a regulatory sequence (i.e., a promoter) is afunctional link that allows for expression of the polynucleotide of theinvention. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions, byoperably linked is intended that the coding regions are in the samereading frame. The cassette may additionally contain at least oneadditional polynucleotide to be cotransformed into the organism.Alternatively, the additional polypeptide(s) can be provided on multipleexpression cassettes. Expression cassettes can be provided with aplurality of restriction sites and/or recombination sites for insertionof the polynucleotide to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containselectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a polynucleotide comprising the silencing elementemployed in the methods and compositions of the invention, and atranscriptional and translational termination region (i.e., terminationregion) functional in plants. The regulatory regions (i.e., promoters,transcriptional regulatory regions, and translational terminationregions) and/or the polynucleotides employed in the invention may benative/analogous to the host cell or to each other. Alternatively, theregulatory regions and/or the polynucleotide employed in the inventionmay be heterologous to the host cell or to each other. As used herein,“heterologous” in reference to a sequence is a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention. For example, a promoter operably linkedto a heterologous polynucleotide is from a species different from thespecies from which the polynucleotide was derived, or, if from thesame/analogous species, one or both are substantially modified fromtheir original form and/or genomic locus, or the promoter is not thenative promoter for the operably linked polynucleotide. As used herein,a chimeric gene comprises a coding sequence operably linked to atranscription initiation region that is heterologous to the codingsequence.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked polynucleotide encodingthe silencing element, may be native with the plant host, or may bederived from another source (i.e., foreign or heterologous) to thepromoter, the polynucleotide comprising silencing element, the planthost, or any combination thereof. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also Guerineauet al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acids Res. 15:9627-9639.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the invention. Thepolynucleotide encoding the silencing element can be combined withconstitutive, tissue-preferred, or other promoters for expression inplants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990)Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol.Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026), and the like. Other constitutive promoters include, forexample, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

An inducible promoter, for instance, a pathogen-inducible promoter couldalso be employed. Such promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. PlantPathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and VanLoon (1985) Plant Mol. Virol. 4:111-116. See also WO 99/43819, hereinincorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-la promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced expressionwithin a particular plant tissue. Tissue-preferred promoters includeYamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997)Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) PlantPhysiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

In one embodiment of this invention the plant-expressed promoter is avascular-specific promoter such as a phloem-specific promoter. A“vascular-specific” promoter, as used herein, is a promoter which is atleast expressed in vascular cells, or a promoter which is preferentiallyexpressed in vascular cells. Expression of a vascular-specific promoterneed not be exclusively in vascular cells, expression in other celltypes or tissues is possible. A “phloem-specific promoter” as usedherein, is a plant-expressible promoter which is at least expressed inphloem cells, or a promoter which is preferentially expressed in phloemcells.

Expression of a phloem-specific promoter need not be exclusively inphloem cells, expression in other cell types or tissues, e.g., xylemtissue, is possible. In one embodiment of this invention, aphloem-specific promoter is a plant-expressible promoter at leastexpressed in phloem cells, wherein the expression in non-phloem cells ismore limited (or absent) compared to the expression in phloem cells.Examples of suitable vascular-specific or phloem-specific promoters inaccordance with this invention include but are not limited to thepromoters selected from the group consisting of: the SCSV3, SCSV4,SCSV5, and SCSV7 promoters (Schunmann et al. (2003) Plant FunctionalBiology 30:453-60; the rolC gene promoter of Agrobacteriumrhizogenes(Kiyokawa et al. (1994) Plant Physiology 104:801-02;Pandolfini et al. (2003) BioMedCentral (BMC) Biotechnology 3:7; Grahamet al. (1997) Plant Mol. Biol. 33:729-35; Guivarc'h et al. (1996); Almonet al. (1997) Plant Physiol. 115:1599-607; the rolA gene promoter ofAgrobacterium rhizogenes (Dehio et al. (1993) Plant Mol. Biol.23:1199-210); the promoter of the Agrobacterium tumefaciens T-DNA gene 5(Korber et al. (1991) EMBO J. 10:3983-91); the rice sucrose synthaseRSs1 gene promoter (Shi et al. (1994) J. Exp. Bot. 45:623-31); the CoYMVor Commelina yellow mottle badnavirus promoter (Medberry et al. (1992)Plant Cell 4:185-92; Zhou et al. (1998) Chin. J. Biotechnol. 14:9-16);the CFDV or coconut foliar decay virus promoter (Rohde et al. (1994)Plant Mol. Biol. 27:623-28; Hehn and Rhode (1998) J. Gen. Virol.79:1495-99); the RTBV or rice tungro bacilliform virus promoter (Yin andBeachy (1995) Plant J. 7:969-80; Yin et al. (1997) Plant J. 12:1179-80);the pea glutamin synthase GS3A gene (Edwards et al. (1990) Proc. Natl.Acad. Sci. USA 87:3459-63; Brears et al. (1991) Plant J. 1:235-44); theinv CD111 and inv CD141 promoters of the potato invertase genes (Hedleyet al. (2000) J. Exp. Botany 51:817-21); the promoter isolated fromArabidopsis shown to have phloem-specific expression in tobacco byKertbundit et al. (1991) Proc. Natl. Acad. Sci. USA 88:5212-16); theVAHOX1 promoter region (Tornero et al. (1996) Plant J. 9:639-48); thepea cell wall invertase gene promoter (Zhang et al. (1996) PlantPhysiol. 112:1111-17); the promoter of the endogenous cotton proteinrelated to chitinase of US published patent application 20030106097, anacid invertase gene promoter from carrot (Ramloch-Lorenz et al. (1993)The Plant J. 4:545-54); the promoter of the sulfate transportergeneSultr1; 3 (Yoshimoto et al. (2003) Plant Physiol. 131:1511-17); apromoter of a sucrose synthase gene (Nolte and Koch (1993) PlantPhysiol. 101:899-905); and the promoter of a tobacco sucrose transportergene (Kuhn et al. (1997) Science 275-1298-1300).

Possible promoters also include the Black Cherry promoter for PrunasinHydrolase (PH DL1.4 PRO) (U.S. Pat. No. 6,797,859), Thioredoxin Hpromoter from cucumber and rice (Fukuda A et al. (2005). Plant CellPhysiol. 46(11):1779-86), Rice (RSs1) (Shi, T. Wang et al. (1994). J.Exp. Bot. 45(274): 623-631) and maize sucrose synthese-1 promoters(Yang., N-S. et al. (1990) PNAS 87:4144-4148), PP2 promoter from pumpkinGuo, H. et al. (2004) Transgenic Research 13:559-566), At SUC2 promoter(Truernit, E. et al. (1995) Planta 196(3):564-70., At SAM-1(S-adenosylmethionine synthetase) (Mijnsbrugge K V. et al. (1996) Planr.Cell. Physiol. 37(8): 1108-1115), and the Rice tungro bacilliform virus(RTBV) promoter (Bhattacharyya-Pakrasi et al. (1993) Plant J.4(1):71-79).

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be used in the present invention.

VI. Compositions Comprising Silencing Elements

One or more of the polynucleotides comprising the silencing element canbe provided as an external composition such as a spray or powder to theplant, plant part, seed, a pest, or an area of cultivation. In anotherexample, a plant is transformed with a DNA construct or expressioncassette for expression of at least one silencing element. In eithercompositions, the silencing element, when ingested by an insect, canreduce the level of a target pest sequence and thereby control the pest(i.e., any pest from the Lepidoptera order, such as, Spodopterafrugiperda). It is recognized that the composition can comprise a cell(such as plant cell or a bacterial cell), in which a polynucleotideencoding the silencing element is stably incorporated into the genomeand operably linked to promoters active in the cell. Compositionscomprising a mixture of cells, some cells expressing at least onesilencing element are also encompassed. In other embodiments,compositions comprising the silencing elements are not contained in acell. In such embodiments, the composition can be applied to an areainhabited by a pest. In one embodiment, the composition is appliedexternally to a plant (i.e., by spraying a field or area of cultivation)to protect the plant from the pest.

In one embodiment, the composition comprising the silencing element thatcontrols a pest from the Lepidoptera order does not comprise aheterologous cationic oligopeptide to facilitate uptake of the RNAi intothe insect cells. Accordingly, in such embodiments, insecticidalactivity occurs in the compositions of the invention (i.e., the plant,plant part, plant cell, or microbe) in the absence of a cationicoligopeptide that is heterologous to the plant, plant part or microbe.The cationic oligopeptide is target non-specific and interactsnon-specifically with RNA via electrostatic interactions andneutralization of charge to penetrate membranes and lacks a specificactivity that promotes a specific interaction with a cell membrane.

The composition of the invention can further be formulated as bait. Inthis embodiment, the compositions comprise a food substance or anattractant which enhances the attractiveness of the composition to thepest.

The composition comprising the silencing element can be formulated in anagriculturally suitable and/or environmentally acceptable carrier. Suchcarriers can be any material that the animal, plant or environment to betreated can tolerate. Furthermore, the carrier must be such that thecomposition remains effective at controlling a pest. Examples of suchcarriers include water, saline, Ringer's solution, dextrose or othersugar solutions, Hank's solution, and other aqueous physiologicallybalanced salt solutions, phosphate buffer, bicarbonate buffer and Trisbuffer. In addition, the composition may include compounds that increasethe half-life of a composition.

It is recognized that the polynucleotides comprising sequences encodingthe silencing element can be used to transform organisms to provide forhost organism production of these components, and subsequent applicationof the host organism to the environment of the target pest(s). Such hostorganisms include baculoviruses, bacteria, and the like. In this manner,the combination of polynucleotides encoding the silencing element may beintroduced via a suitable vector into a microbial host, and said hostapplied to the environment, or to plants or animals.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may be stablyincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon,or transiently expressed (e.g., transfected mRNA).

Microbial hosts that are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops ofinterest may be selected. These microorganisms are selected so as to becapable of successfully competing in the particular environment with thewild-type microorganisms, provide for stable maintenance and expressionof the sequences encoding the silencing element, and desirably, providefor improved protection of the components from environmental degradationand inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum,Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris,Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli andAzotobacter vinlandir, and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans. Of particular interest are the pigmentedmicroorganisms.

A number of ways are available for introducing the polynucleotidecomprising the silencing element into the microbial host underconditions that allow for stable maintenance and expression of suchnucleotide encoding sequences. For example, expression cassettes can beconstructed which include the nucleotide constructs of interest operablylinked with the transcriptional and translational regulatory signals forexpression of the nucleotide constructs, and a nucleotide sequencehomologous with a sequence in the host organism, whereby integrationwill occur, and/or a replication system that is functional in the host,whereby integration or stable maintenance will occur.

Transcriptional and translational regulatory signals include, but arenot limited to, promoters, transcriptional initiation start sites,operators, activators, enhancers, other regulatory elements, ribosomalbinding sites, an initiation codon, termination signals, and the like.See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2;Sambrook et al. (2000); Molecular Cloning: A Laboratory Manual (3rd ed.;Cold Spring Harbor Laboratory Press, Plainview, N.Y.); Davis et al.(1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.); and the references cited therein.

Suitable host cells include the prokaryotes and the lower eukaryotes,such as fungi. Illustrative prokaryotes, both Gram-negative andGram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia,Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such asRhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia,Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae;Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceaeand Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetesand Ascomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of the invention include ease of introducing the codingsequence into the host, availability of expression systems, efficiencyof expression, stability in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities, such as thick cell walls,pigmentation, and intracellular packaging or formation of inclusionbodies; leaf affinity; lack of mammalian toxicity; attractiveness topests for ingestion; and the like. Other considerations include ease offormulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorulaspp., Aureobasidium spp., Saccharomyces spp., and Sporobolomyces spp.,phylloplane organisms such as Pseudomonas spp., Erwinia spp., andFlavobacterium spp., and other such organisms, including Pseudomonasaeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillusthuringiensis, Escherichia coli, Bacillus subtilis, and the like.

The sequences encoding the silencing elements encompassed by theinvention can be introduced into microorganisms that multiply on plants(epiphytes) to deliver these components to potential target pests.Epiphytes, for example, can be gram-positive or gram-negative bacteria.

The silencing element can be fermented in a bacterial host and theresulting bacteria processed and used as a microbial spray in the samemanner that Bacillus thuringiensis strains have been used asinsecticidal sprays. Any suitable microorganism can be used for thispurpose. Pseudomonas has been used to express Bacillus thuringiensisendotoxins as encapsulated proteins and the resulting cells processedand sprayed as an insecticide Gaertner et al. (1993), in AdvancedEngineered Pesticides, ed. L. Kim (Marcel Decker, Inc.).

Alternatively, the components of the invention are produced byintroducing heterologous genes into a cellular host. Expression of theheterologous sequences results, directly or indirectly, in theintracellular production of the silencing element. These compositionsmay then be formulated in accordance with conventional techniques forapplication to the environment hosting a target pest, e.g., soil, water,and foliage of plants. See, for example, EPA 0192319, and the referencescited therein.

In the present invention, a transformed microorganism can be formulatedwith an acceptable carrier into separate or combined compositions thatare, for example, a suspension, a solution, an emulsion, a dustingpowder, a dispersible granule, a wettable powder, and an emulsifiableconcentrate, an aerosol, an impregnated granule, an adjuvant, a coatablepaste, and also encapsulations in, for example, polymer substances.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients of the present invention (i.e., at least one silencingelement) are normally applied in the form of compositions and can beapplied to the crop area, plant, or seed to be treated. For example, thecompositions may be applied to grain in preparation for or duringstorage in a grain bin or silo, etc. The compositions may be appliedsimultaneously or in succession with other compounds. Methods ofapplying an active ingredient or a composition that contains at leastone silencing element include, but are not limited to, foliarapplication, seed coating, and soil application. The number ofapplications and the rate of application depend on the intensity ofinfestation by the corresponding pest.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; carboxylate ofa long chain fatty acid; an N-acylsarcosinate; mono- or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitan fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include, but are not limited to, inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions comprising the silencing element can be in a suitableform for direct application or as a concentrate of primary compositionthat requires dilution with a suitable quantity of water or otherdilutant before application.

The compositions (including the transformed microorganisms) can beapplied to the environment of an insect pest (such as a pest from theLepidoptera order) by, for example, spraying, atomizing, dusting,scattering, coating or pouring, introducing into or on the soil,introducing into irrigation water, by seed treatment or generalapplication or dusting at the time when the pest has begun to appear orbefore the appearance of pests as a protective measure. For example, thecomposition(s) and/or transformed microorganism(s) may be mixed withgrain to protect the grain during storage. It is generally important toobtain good control of pests in the early stages of plant growth, asthis is the time when the plant can be most severely damaged. Thecompositions can conveniently contain another insecticide if this isthought necessary. In an embodiment of the invention, the composition(s)is applied directly to the soil, at a time of planting, in granular formof a composition of a carrier and dead cells of a Bacillus strain ortransformed microorganism of the invention. Another embodiment is agranular form of a composition comprising an agrochemical such as, forexample, a herbicide, an insecticide, a fertilizer, in an inert carrier,and dead cells of a Bacillus strain or transformed microorganism of theinvention.

VII. Plants, Plant Parts, and Methods of Introducing Sequences intoPlants

In one embodiment, the methods of the invention involve introducing apolypeptide or polynucleotide into a plant. “Introducing” is intended tomean presenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the invention do not depend on a particular methodfor introducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotide or polypeptides intoplants are known in the art including, but not limited to, stabletransformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, U.S. Pat. No.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. Nos. 5,886,244; and,5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation(WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783;and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize);Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-VanSlogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the silencing element sequences of theinvention can be provided to a plant using a variety of transienttransformation methods. Such transient transformation methods include,but are not limited to, the introduction of the protein or variants andfragments thereof directly into the plant or the introduction of thetranscript into the plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway etal. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci.44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 andHush et al. (1994) The Journal of Cell Science 107:775-784, all of whichare herein incorporated by reference. Alternatively, polynucleotides canbe transiently transformed into the plant using techniques known in theart. Such techniques include viral vector system and the precipitationof the polynucleotide in a manner that precludes subsequent release ofthe DNA. Thus, the transcription from the particle-bound DNA can occur,but the frequency with which it is released to become integrated intothe genome is greatly reduced. Such methods include the use of particlescoated with polyethylimine (PEI; Sigma #P3143).

In other embodiments, the polynucleotide of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. Further, it is recognized that promoters of the invention alsoencompass promoters utilized for transcription by viral RNA polymerases.Methods for introducing polynucleotides into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) MolecularBiotechnology 5:209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species of interest include, but are not limited to, corn (Zeamays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularlythose Brassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsare optimal, and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

VIII. Methods of Use

The methods of the invention comprise methods for controlling a pest(i.e., pest from the Lepidoptera order, such as, Spodoptera frugiperda).The method comprises feeding to a pest a composition comprising asilencing element of the invention, wherein said silencing element, wheningested by a pest (i.e., pests from the Lepidoptera order, such as,Spodoptera frugiperda), reduces the level of a target polynucleotide ofthe pest and thereby controls the pest. The pest can be fed thesilencing element in a variety of ways. For example, in one embodiment,the polynucleotide comprising the silencing element is introduced into aplant. As the Lepidoptera feeds on the plant or part thereof expressingthese sequences, the silencing element is delivered to the pest. Whenthe silencing element is delivered to the plant in this manner, it isrecognized that the silencing element can be expressed constitutively oralternatively, it may be produced in a stage-specific manner byemploying the various inducible or tissue-preferred or developmentallyregulated promoters that are discussed elsewhere herein. In specificembodiments, the silencing element expressed in the roots, stalk orstem, leaf including pedicel, xylem and phloem, fruit or reproductivetissue, silk, flowers and all parts therein or any combination thereof.

In another method, a composition comprising at least one silencingelement of the invention is applied to a plant. In such embodiments, thesilencing element can be formulated in an agronomically suitable and/orenvironmentally acceptable carrier, which is preferably, suitable fordispersal in fields. In addition, the carrier can also include compoundsthat increase the half life of the composition. In specific embodiments,the composition comprising the silencing element is formulated in such amanner such that it persists in the environment for a length of timesufficient to allow it to be delivered to a pest. In such embodiments,the composition can be applied to an area inhabited by a pest. In oneembodiment, the composition is applied externally to a plant (i.e., byspraying a field) to protect the plant from pests.

In certain embodiments, the constructs of the present invention can bestacked with any combination of polynucleotide sequences of interest inorder to create plants with a desired trait. A trait, as used herein,refers to the phenotype derived from a particular sequence or groups ofsequences. For example, the polynucleotides of the present invention maybe stacked with any other polynucleotides encoding polypeptides havingpesticidal and/or insecticidal activity, such as other Bacillusthuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986)Gene 48:109), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825,pentin (described in U.S. Pat. No. 5,981,722), and the like. Thecombinations generated can also include multiple copies of any one ofthe polynucleotides of interest. The polynucleotides of the presentinvention can also be stacked with any other gene or combination ofgenes to produce plants with a variety of desired trait combinationsincluding, but not limited to, traits desirable for animal feed such ashigh oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids(e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802;and 5,703,409); barley high lysine (Williamson et al. (1987) Eur. J.Biochem. 165:99-106; and WO 98/20122) and high methionine proteins(Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988)Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12:123));increased digestibility (e.g., modified storage proteins (U.S. Pat. No.6,858,778); and thioredoxins (U.S. Pat. No. 7,009,087)); the disclosuresof which are herein incorporated by reference.

The polynucleotides of the present invention can also be stacked withtraits desirable for disease or herbicide resistance (e.g., fumonisindetoxification genes (U.S. Pat. No. 5,792,931); avirulence and diseaseresistance genes (Jones et al. (1994) Science 266:789; Martin et al.(1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS gene)); and traits desirable for processing or processproducts such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils(e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase),starch synthases (SS), starch branching enzymes (SBE), and starchdebranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S.Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, andacetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs));the disclosures of which are herein incorporated by reference. One couldalso combine the polynucleotides of the present invention withpolynucleotides providing agronomic traits such as male sterility (e.g.,see U.S. Pat. No. 5,583,210), stalk strength, flowering time, ortransformation technology traits such as cell cycle regulation or genetargeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821); thedisclosures of which are herein incorporated by reference.

These stacked combinations can be created by any method including, butnot limited to, cross-breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

Methods and compositions are further provided which allow for anincrease in RNAi produced from the silencing element. In suchembodiments, the methods and compositions employ a first polynucleotidecomprising a silencing element for a target pest sequence operablylinked to a promoter active in the plant cell; and, a secondpolynucleotide comprising a suppressor enhancer element comprising thetarget pest sequence or an active variant or fragment thereof operablylinked to a promoter active in the plant cell. The combined expressionof the silencing element with suppressor enhancer element leads to anincreased amplification of the inhibitory RNA produced from thesilencing element over that achievable with only the expression of thesilencing element alone. In addition to the increased amplification ofthe specific RNAi species itself, the methods and compositions furtherallow for the production of a diverse population of RNAi species thatcan enhance the effectiveness of disrupting target gene expression. Assuch, when the suppressor enhancer element is expressed in a plant cellin combination with the silencing element, the methods and compositioncan allow for the systemic production of RNAi throughout the plant; theproduction of greater amounts of RNAi than would be observed with justthe silencing element construct alone; and, the improved loading of RNAiinto the phloem of the plant, thus providing better control of phloemfeeding insects by an RNAi approach. Thus, the various methods andcompositions provide improved methods for the delivery of inhibitory RNAto the target organism. See, for example, U.S. Provisional ApplicationNo. 61/021,676, entitled “Compositions and Methods for the Suppressionof Target Polynucleotides”, filed Jan. 17, 2008 and herein incorporatedby reference in its entirety.

As used herein, a “suppressor enhancer element” comprises apolynucleotide comprising the target sequence to be suppressed or anactive fragment or variant thereof. It is recognize that the suppressorenhancer element need not be identical to the target sequence, butrather, the suppressor enhancer element can comprise a variant of thetarget sequence, so long as the suppressor enhancer element hassufficient sequence identity to the target sequence to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element. Similarly,the suppressor enhancer element can comprise a fragment of the targetsequence, wherein the fragment is of sufficient length to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element. Thus, inspecific embodiments, the suppressor enhancer element comprises afragment or a variant of a polynucleotide encoding a juvenile hormonepolypeptide, a vacuolar polypeptide, a cadherin polypeptide, a cuticlepolypeptide, a translation initiation factor, a SARI polypeptide, anelongation factor, a phosphooligosaccharide, a myosin polypeptide, apotassium channel amino acid transporter, a potassium inwardly rectifierpolypeptide, an amino acid transporter, a tubulin polypeptide, aubiquitin polypeptide, small nuclear ribonucleoprotein, or any otherpolynucleotide of interest disclosed herein. In still other embodiments,the suppressor enhancer element comprises a polynucleotide set forth inSEQ ID NO: 1-50 or an active variant or fragment thereof.

It is recognized that multiple suppressor enhancer elements from thesame target sequence or from different target sequences, or fromdifferent regions of the same target sequence can be employed. Forexample, the suppressor enhancer elements employed can comprisefragments of the target sequence derived from different region of thetarget sequence (i.e., from the 3′UTR, coding sequence, intron, and/or5′UTR). Further, the suppressor enhancer element can be contained in anexpression cassette, as described elsewhere herein, and in specificembodiments, the suppressor enhancer element is on the same or on adifferent DNA vector or construct as the silencing element. Thesuppressor enhancer element can be operably linked to a promoter asdisclosed herein. It is recognized that the suppressor enhancer elementcan be expressed constitutively or alternatively, it may be produced ina stage-specific manner employing the various inducible ortissue-preferred or developmentally regulated promoters that arediscussed elsewhere herein.

In specific embodiments, employing both a silencing element and thesuppressor enhancer element the systemic production of RNAi occursthroughout the entire plant. In further embodiments, the plant or plantparts of the invention have an improved loading of RNAi into the phloemof the plant than would be observed with the expression of the silencingelement construct alone and, thus provide better control of phloemfeeding insects by an RNAi approach.

In specific embodiments, the plants, plant parts, and plant cells of theinvention can further be characterized as allowing for the production ofa diversity of RNAi species that can enhance the effectiveness ofdisrupting target gene expression.

In specific embodiments, the combined expression of the silencingelement and the suppressor enhancer element increases the concentrationof the inhibitory RNA in the plant cell, plant, plant part, plant tissueor phloem over the level that is achieved when the silencing element isexpressed alone.

As used herein, an “increased level of inhibitory RNA” comprises anystatistically significant increase in the level of RNAi produced in aplant having the combined expression when compared to an appropriatecontrol plant. For example, an increase in the level of RNAi in theplant, plant part or the plant cell can comprise at least about a 1%,about a 1%-5%, about a 5%-10%, about a 10%-20%, about a 20%-30%, about a30%-40%, about a 40%-50%, about a 50%-60%, about 60-70%, about 70%-80%,about a 80%-90%, about a 90%-100% or greater increase in the level ofRNAi in the plant, plant part, plant cell, or phloem when compared to anappropriate control. In other embodiments, the increase in the level ofRNAi in the plant, plant part, plant cell, or phloem can comprise atleast about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold,about a 10 fold-20 fold, about a 20 fold-30 fold, about a 30 fold-40fold, about a 40 fold-50 fold, about a 50 fold-60 fold, about 60 fold-70fold, about 70 fold-80 fold, about a 80 fold-90 fold, about a 90fold-100 fold or greater increase in the level of RNAi in the plant,plant part, plant cell or phloem when compared to an appropriatecontrol. Methods to assay for an increase in the level of RNAi arediscussed elsewhere herein.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Specific Target Genes and Silencing Elements thatCause Insecticidal Activity Against Spodoptera Frugiperda

Disruption of insect gene function via RNAi can produce specificactivity against target insects. This specificity is enhanced bydelivery of the dsRNAs via transgenic plants. Identification of genefunction in insects via RNAi has been largely limited to injection ofdsRNAs. In fact, past experiments have indicated that insects are notcapable of systemic RNAi response based on exposure to dsRNAs.

As described below, we have demonstrated acute activity of numerousdsRNA pairs through injection experiments and additionally havedemonstrated insect antagonism through ingestion of dsRNAs. Thisevidence identifies several gene/primer pair combinations with clearinsecticidal properties. The use of dsRNAs in transgenic plants alsoaddresses the potential complication of heterologous protein expressionand the possible risks of allergic reaction, non-target activity, andenvironmental- or bioaccumulation. The data presented below representsthe first test of disruption of these particular genes resulting ininsecticidal activity in whole organisms and the first report ofinsecticidal activity of dsRNAs against Spodoptera frugiperda.

The invention describes specific target genes and the dsRNA sequencescausing insecticidal activity against the Lepidopteran Spodopterafrugiperda through RNA interference of the target gene's expression.Disruption of the genes targeted by the dsRNA sequences may be broadlyinsecticidal in numerous species. The specific dsRNA sequences displayinsecticidal activity upon ingestion and can be utilized with atransgenic plant mode of delivery. Table 1 provides the polynucleotideof non-limiting examples of target sequence from Spodoptera frugiperda,a brief description of the function of the protein encoded by the targetsequence, and a SEQ ID NO. Table 2 provides a summary of primers used tosuppress the target polynucleotides.

TABLE 1Target Polynucleotides from Spodoptera frugiperda. >ise1c.pk002.m13 Juvenile hormone querySEQ ID NO: 1CAAGCATCCAACATGGTATCCGACTTCAGGAAGAAGAAGCTCCTCCACGTGTTCAAGTCCTTCTTCGACACGGACGGCAGCGGCAACATCGAGAAGGATGACTTCCTGATGGCCATCGAAAGGATAACCAAGACCAGAGGCTGGAAAGCTGGAGACGACAAATACAAATTTGTCGAGGAGACCCTATTGAAGATCTGGGACGGCATCCAGAAGGTCGCTGACGAGAACAAGGACGGACAGGTCAGCCAGGACGAGTGGATCGCTATGTGGGACAAGTACTCCAAGAACCCGTCCGAGGCGTTCGAGTGGCAGACCCTGTACTGCAAGTTCGCGTTCACTCTTGAAGACGCCAGCGACGATGGATCCATCGACAGCGAGGAGTTCTCCTCTGTGTACGCCTCCTTCGGCCTGGACAANGACGANGCTGTGGCTGCCTTCAAGAAAGATGGCTAACGGTAAGTCCGAAGTGTCCTGGGCTTGAGTTCCACGACCTGTGGAANGAGTACTTCTCATCCGGAAGACTNGAACGCTGCCGGCAAN >ise1c.pk003.f7 Juvenile hormone query SEQ ID NO: 2CCAACATGGTATCCGACTTCAGGAAGAAGAAGCTCCTCCACGTGTTCAAGTCCTTCTTCGACACGGACGGCAGCGGCAACATCGAGAAGGATGATTTCCTGATGGCCATCGAAAGGATAACCAAGACCAGAGGCTGGAAAGCTGGAGACAACAAATACAAATTTGTCGAGGAAACCCTATTGAAGATCTGGGACGGCATCCAGAAGGTCGCTGACGAGAACAAGGACGGACAGGTCAGCCAGGACGAGTGGATCGCTATGTGGGACAAGTACTCCAAGAACCCATCCGAGGCGTTCGAGTGGCAGACCCTGTACTGCAAGTTCGCGTTCACTCTTGAAGACGCCAGCGACGACGGATNCATCGACAGCGAAGAGTTCTCCTCTGTGTACGCCTCCTTCGGGCTGGACAANGGACGAGGCGGTGGCTGCCTTCAAGAAGATGNTAACGGTAAGTCCGAATGTCCTGGGGCTGAGTTTCAAGANCTGTTGGAAGGATACTTCTCAAC >ise1c.pk005.a15 Juvenile hormone querySEQ ID NO: 3CAACATGGTATCCGACTTCAGGAAGAATAAGCTCCTCCACGTGTTCAAGTCCTTCTTCGACACGGACGGCAGCGGCAACATCGAGAAGGATGACTTCCTGATGGCCATCGAAAGGATAACCAAGACCAGAGGCTGGAAAGCTGGAGACGACAAATACAAATTTGTCGAGGAGACCCTATTGAAGATCTGGGACGGCATCCAGAAGGTCGCTGACGAGAACAAGGACGGACAGGTCAGCCAGGACGAGTGGATCGCTATGTGGGACAAGTANTCCAAGAACCCGTCCGAGGCGTTCGAGTGGCAGACCCTGTACTGCAAGTTCGCGTTCACTCTTGAAGACGCCAGNGACGATGGATCCATCGACAGCGAGGAGTTCTCCTCTGTGTACGCCTCCTTCGGCCTGGACAAGG >ise1c.pk006.d24 Juvenile hormone querySEQ ID NO: 4GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTNGGAAANTACTATTTTATTGTACCAACTGCCCCTTAACCTCATCTATGAGTCACCCATAAATGTTATTTTGGTAAAATGTTTGACACACTTCACACTAATATTTATAAATGTGAAAGTTTGTTTGTTTGAATGTTTGTATATTTGTCTGTCAATCACGCTGAAACCACTGTATAGAATTTGACCTAATTTGGTATACANACAGGGTATGAGCTGACTTGGGTGATAGGATACTTTTTATCCCACAGGAACGCGGGTAAAGTCCNTGGGCAGAAGCTAGTATGTAATAATTATNTCCCTCTACCTACCCTATATGGGGGTGGACCGTCATGTTCTTTACNCNACAACCNGTTTGTCCACCTCNCCTTTAAAGTTTTGTNAG >ise2c.pk009.i4 Juvenile hormone querySEQ ID NO: 5GCACGAGGGCCGTGTCGACTTCGCACCAGTCCCCTATTTATTTACCTTGACAAAAATATGGCGCGCCTATTGTTTATTGCGCCTATCCTGGCGTTGGCTATAATGCCAGTATACTTCTTATTCCTAAAGGGACCACCCCCACTACCCGAACTAGATATGAACGAGTGGTGGGGCCCAGAGAAGCTAAAAGCAAAACCTGACACTAGTATAAAACCCTTTAAAATTGCTTTTGGAGACACTGTTGTAAAAGACTTAAAAGACCGTCTCAAACGTTCTCGGTCTTTCACTGCTCCGCTGGAGGGTGTGGCATTCCAGTACGGCTTCAACACTGCTCAGCTGGATGGTTGGCTGAAGTACTGGGCTAATGAGTATAAGTTCAAGGAGAGAGAGACCTTCCTCAACCAGTACCCTCAGTACAAAACCAATATCCAGGGTCTTGACATCCACTTCATCAGGGTTACACCGAAGGTACCGGCAGGAGTGGAGGTGGTACCCATGCTACTCCTCCACGGCTGGCCAGGCTCTGTCAGGGAGTTCTACGAGGCTATTCCTCTCATCACAGCAGTCAGCAAGGACCGTGACTTCGCTGTGGAAGTCATCGTTCCAAGTCTACCTGGCTATGGATTCTCTGATGCCGCAGTTCGTCCCGGCnnnnnnnCCCCACAAATGnnn >ise2c.pk001.d19 vacuolar querySEQ ID NO: 6GCACGAGGCTTGGACGTGATGTTACCTGGGAATTCAACCCCTTGAATGTTAAGGTCGGCTCCCACATCACCGGAGGAGACTTGTACGGTATCGTACACGAGAACACATTGGTTAAGCACAAGATGTTGATCCCACCCAAGGCCAAGGGTACCGTCACCTACGTCGCGCCCTCCGGCAACTACAAAGTCACTGACGTAGTGTTGGAGACGGAGTTCGACGGCGAGAAGGAGAAGTACACCATGTTGCAAGTATGGCCGGTGCGCCAGCCGCGCCCCGTCACTGAGAAGCTGTCCGCCAACCACCCCCTGCTCACCGGACAGAGAGTGCTCGACTCTCTCTTCCCTTGTGTCCAGGGTGGTACCACGGCCATCCCCGGCGCCTTCGGTTGTGGCAAGACTGTCGTCTCACAGGCTCTGTCCAAGTACTCCAACTCTGACGTCATCATCTACGTCGGATGCGGTGAACGTGGTAACGAGATGTCTGAGGTACTGCGTGACTTCCCCGAGCTGACGGTGGAGATCGAGGGCATGACCGAGTCCATCATGAAGCGTACCGCGCTCGTCGCCAACACCTCCAACATGCCTGTAGCCGCCCGAGAGGCTTCCATCTACACCGGTATCACCCTCTCCGAGTACTTCCGTGACATGGGTTACAACGTGTCCATGATGGCTGACTCCACCTCTCGTTGGGCCG >ise2c.pk001.e14 vacuolar query SEQ ID NO: 7GCACGAGGCAGATAGTCATCACTGTTTTTGGGACCTGTnnnTACTCCCTCAATAAACCTACAAAATGGCCGAAAACCCAATCTACGGACCCTTCTTTGGAGTTATGGGGGCGGCGTCTGCTATCATCTTTAGCGCGCTGGGAGCTGCCTATGGAACTGCTAnGnCnnnnACCGGTATCGCCGCCATGTCGGTGATGCGGCCCGAGCTCATCATGAAGTCCAACAACTACACCCTTTACAAGnGGTTCATCCACCTTGGCGCTGGTCTnnnCGTAAGTTTCTCCGGTCTAGCGnnnGGCnn >ise2c.pk001.f20 vacuolar querySEQ ID NO: 8GCACGAGGCTCACAGGCTCTGTCCAAGTACTCCAACTCTGACGTCATCATCTACGTCGGATGCGGTGAACGTGGTAACGAGATGTCTGAGGTACTGCGTGACTTCCCCGAGCTGACGGTGGAGATCGAGGGCATGACCGAGTCCATCATGAAGCGTACCGCGCTCGTCGCCAACACCTCCAACATGCCTGTAGCCGCCCGAGAGGCTTCCATCTACACCGGTATCACCCTCTCCGAGTACTTCCGTGACATGGGTTACAACGTGTCCATGATGGCTGACTCCACCTCTCGTTGGGCCGAGGCTCTTCGTGAGATCTCnnnnCGTCTGGCTGAGATGCCTGCCGACTCGGGTTACCCCGCCTACCTGGGAGCCCGTCTGGCCTCGTTCTACGAGCGTGCCGGACGTGTGAAGTGCTTGGGTAACCCCGACAGGGAGGGCTCCGTGTCCATCGTGGGCGCCGTGTCGCCGCCCGGAGGTGACTTCTCCGACCCCGTGACGGCCGCCACGCTGGGTATCGTGCAGGTGTTCTGGGGGTTGGACAAGAAGCTCGCGCAGCGCAAGCACTTCCCCGCCATCAACTGGCTCATCTCCTACAGCAAGTACATGCGAGCGCTGGACGACTTCTATGAGAAGAACTACCCCGAGTTCGTGCCCCTCnnnnnCAAGGGTCAAGGAGATCCTGCAGnnn >ise2c.pk010.h3 cadherin query SEQ ID NO: 9GCACGAGGTATCTAAAACAGTGCGTCGTAATATATTCAAGATGTCTCGTCTTAGGTTTTGTTTTTTATTAGCAGTACTATGCAGTTGTTTGCAGAATGGTTACGGTTTTACAACAGAAAAGCCAGTTACCCAGCATGTAGATCCTAAACCAGAAGTTCCTGAAACGTTGCCTGAAACAACACGAGTGCCTGCGCCGAGCTCGTCGACGGCAGCGCCGACCACACCAGCTCCGACACCGGCACCAACGCCAGCACCCACACCAGCTCCTACACCAGCTCCTACTCCAGCTCCTACCCCTGCGCCTACTCCTGCGCCTACTCCTGCGCCTACTCCTGCGCCTACCCCCGCACCTACACCAGCGCCCACTCCTGCTCCCACCCCAGCTCCCCTCCCCGCCCCCGACCAAGGCACATGGTCCTTCACTGATGAAAAGGCCAATCAGACATGCATTGTGGCCCAATTCGCAGCCCAACTGAATGTCACATACACCAAGTTAGTGGAGAATGCAACGTCTCTATCGTACGTGAGGCTCAACGTGCCCGCGAACGCGTCGGTCCTCAACGGCAGCTGTTCGGACCCCGACCAATGGATCCAGATCACCTGGAAGACCAACGACGACAGCGAGACGAACAACACCATGACCCTCGTGTACAACAAGAATGCCACCACCAAGnnCTACGGCCTG >ise2c.pk011.a10 cuticle protein SEQ ID NO: 10GCACGAGGGCGGTTTGAAGTGATCTAGTTCGTCAGAAAAAACACAGACCACGTTCACAATGAAATCGATGGTGGTGTTATTCGCTGTGTGCGCCGTGGCGTGCGGCTCCCTGGTGCCGCTGGCGCAGCCTCCTCATCACCCCGCCGTCGTGCTGGACCCGCACGGCCGCCCGCTCGACACCGCCGAGGTGATCAACGCCCGCGCCCTCCACCTGCAGGCTAAGGCCCTGGATGGACACTACGCTCCCCTCGCGCACGCTGCCGTCGTGCCTGTTGCCCACTCCGTGGTAGCCGCCCCCGCTGTGGTCGCCGCTCCCGCCGCCGTGTCCCACCAGTCCCGTGTGGATGTGCGCACCAGCCCCGCCATCGTGAGCCACGCCGTCGCTGCTCCCGTAGTAGCCCACGGTGTCTACTCCGCTCCCCTGCTGGCCCACTCCGCTCTCGGCTACGCCGGTCACGGACACTACCTGAAGAAGCGCTCCCTGGGACACCTCGCCTACGCCGCTCCCGTCGTCGCCCACGTAGCTCCCTCCGCGGTGTCGCACCAGTCCCGCGTGGnCnTCGTCTCCAGCCnnnCTGTCGTGTCTCAnnnnnTnnnTnCCGTnnTGTCCCn >ise2c.pk011.h12 cuticle protein SEQ ID NO: 11GCACGAGGGGACGTTGAACGAAAGAAAATGCTACGCGTTACGATTTTAGCCGCAGTGGTGGTGTTCGCCTCAGGCGCGCCCCAGAACAACTTCATCTTCAAGAATGACATCACTCCTGAGGAAGCCCAGCAGTACCTCAAACAACTGCCGTTCACCTCACCCCAGCTCTCTGGACGCACCGCTGTACTGCCTCTGGTTCGCTACGACGACCCCAGGTTTCGTTCAGCTGAAGCTGGCCCAACCCTTGGACACTACTGGAAGAATGGACAGGAGATCCAGAACACAGAGGACTACTTAGAAGAGGTCTACAACGCGGCTCAATACCACGGCCAGGACGGTCTTGGCAACTACGCCTACGGTTATGAGACCCCTGAATCTTCCAAGGTTGAGAACCGTGAAGGTTCCGGAGTCGTCCAAGGATCCTATGTGTACCAGGTTCCCGGAATGAAGGATCTCGTCnnGGTCCGTTACTGGGCTGACAGCCnnnnnTTCCACCAGnAnGACAATCTTCCCAAGGTTGAACTGAnnnCCGCTnnnnnnnnCCCGCTCT >ise2c.pk001.d22 translation initiation factorSEQ ID NO: 12GCACGAGGTATCACTCCTGACCGTATCTAAAACTCGGCACACAACACAATGGCTGACATCGAAGATACACATTTCGAGACCGGGGACTCCGGTGCCTCCGCCACCTTCCCTATGCAATGCTCGGCCCTGCGCAAGAACGGTTTCGTCATGCTTAAGGGTCGCCCCTGCAAAATCGTCGAGATGTCCACTTCCAAAACCGGAAAGCACGGCCACGCTAAAGTTCACTTGGTTGGAATCGATATTTTTAACGGCAAGAAATACGAAGATATCTGCCCTTCCACCCACnnnCATGGACGTGCCCCACGTGAAGCGTGAGGACTACCAGCTCACCGATATCTCTGACGACGGCTACCTTACCCTCATGGCTGACAACGGCGATCTCCGCGAGGACCTCAAGATCCCAGACGGTGACCTCGGCACCCAGTTGCGTTCTGACTTCGATAGCGGCAAAGAGCTGTTGTGCACTGTGCTGAAGTCTTGCGGTGAGGAGTGTGTAATCGCAGTCAAGGCAAACACAGCTCTCGACAAATAAACCAACTCAGCATTTATAGGGATATACATACATATAATTTTTTTACAATCAACAGCTCTTACATAAATGTAAAACATAATACTATGTATAATTTAACATnnnnnATTATGGTGTGACGCGGTGCTGGCTTGTCGCCGTCCACTCCACCCCCGAAG >ise2c.pk001.d9 translation initiation factor SEQ ID NO: 13GCACGAGGCGCGATTGTAACATGTCGTATTCACCAGAAAGAAGATCAGAAGATTGGCCGGAAGATTCCAAAAATGGCCCGTCTAAGGATCAAGGCAACTATGATGGGCCTCCAGGAATGGAACCCCAAGGGGCACTTGATACAAACTGGCACCAGGTCGTGGAAAGCTTTGACGACATGAATCTGAAGGAAGAATTGTTGAGAGGAATTTATGCTTACGGTTTTGAAAAGCCGTCTGCTATCCAACAACGCGCTATTATGCCTTGCATTCAAGGCCGTGATGTCATAGCTCAAGCCCAGTCTGGTACTGGGAAGACTGCTACCTTCTCTATTTCAATTCTTCAGCAAATCGATACCAGTATTCGTGAATGCCAAGCACTGATTTTGGCCCCTACTAGAGAGCTGGCTCAGCAGATCCAAAAGGTGGTGATTGCTCTTGGGGATCACTTGAATGCTAAATGCCATGCTTGCATCGGCGGCACTAATnnnGCGCGAAGATGTTCGTCAGCTnnnnn >ise2c.pk001.i23 translation initiation factorSEQ ID NO: 14GCACGAGGGTCGTATTCACCAGAAAGAAGATCAGAAGATTGGCCGGAAGATTCCAAAAATGGCCCGTCTAAGGATCAAGGCAACTATGATGGGCCTCCAGGAATGGAACCCCAAGGGGCACTTGATACAAACTGGCACCAGGTCGTGGAAAGCTTCGACGACATGAATCTGAAGGAAGAATTGTTGAGAGGAATTTATGCTTACGGTTTTGAAAAGCCGTCTGCTATCCAACAACGCGCTATTATGCCTTGCATTCAAGGCCGTGATGTCATAGCTCAAGCCCAGTCTGGTACTGGGAAGACTGCTACCTTCTCTATTTCAATTCTTCAGCAAATCGATACCAGTATTCGTGAATGCCAAGCACTGATTTTGGCCCCTACTAGAGAGCTGGCTCAGCAGATCCAAAAGGTGGTGATTGCTCTTGGGGATCACTTGAATGCTAAATGCCATGCTTGCATCGGCGGCACTAATGTGCGCGAAGATGTTCGTCAGCTGGAGAGTGGTGTGCATGTGGTGGTGGGTACACCTGGTCGCGTGTACGACATGATAACTCGTCGTGCTCTCCGTGCTAACACTATCAAGCTGTTTGTACTTGATGAAGCTGATGAAATGCTGTCAAGAGGATTTAAAGATCnn >ise2c.pk001.l24 translation initiation factorSEQ ID NO: 15GCACGAGGGCCATCCTGTCACACATCTACCACCACGCCCTGCACGATAACTGGTTCCAAGCTCGAGACTTGCTCTTGATGTCACACTTGCAAGAGACTGTTCAACATTCAGACCCGAGCACTCAGATTTTGTACAATCGTACTATGGCCAATCTAGGTTTGTGCGCTTTTCGAAGGGGCAATGTTAAAGAAGCCCATGGCTGCCTAGCTGAACTGATGATGACTGGCAAACCCAAGGAACTGTTAGCTCAAGGTCTGCTACCTCAGCGTCAACACGAGCGTTCAAAGGAACAGGAAAAGATAGAGAAGCAACGCCAAATGCCGTTCCACATGCACATCAACTTGGAACTGCTTGAATGTGTGTATTTAGTGTCTGCCATGCTGATTGAAATTCCATACATGGCCGCCCACGAATTCGATGCTCGCCGGCGCATGATTAGTAAGACTTTCTATCAGAATTTGCGCGCAAGTGAGCGTCAGGCTTTGGTAGGCCCGCCCGAATCCATGCGTGAGCATGCTGTGGCTGCCGCCAGGGCGATGCGCCGCGGAGACTGGCGTGCTTGCCTCAATTTTATTGTnnnTGnnnAATGAAT >ise2c.pk005.b9 translation initiation factorSEQ ID NO: 16GCACGAGGCTGATAGCCACCTGCCAAATTATCTTGAAATATAACCATTCACTAAAATATTTAACGTAATTTAGTGGTTAATTCTAAACTTAATCATGGACGACGACATGGTATTTGATCCATCTTTAAAGAAAAAGAAGAAGAAGAAGACCGGTTTCGACTTAGATGCCGCTCTCGCAGGCGAACAAGGTGAGAGCACGAGCGTGGAGGCGCCCGCTGGGTCGGGTGACGTCGACTTGCCTGAGGATGATAACCTCGATTTGGATAATTTTGGAAAGAAAAAGAAGAAGAAGAAGAAGGGAGTCTTCAACATGGAAGAACTTGAAAGTACGTTACCGGAAACACCTCCGGCCGAAGAGCCGGAACAGCAGGAGGACGAAGTTATTGACGATTTAGATCTAGATATTGACTTCTCTAAAACGAAAAAGAAGAAGAAGAAGAAAAACATnnnAnGAGCTCGTCCTTGAAGATGACACCAAGGGAGAAGATCAAGAGAATGTCGAGGATGTTAGTGGTGATTTATGGAGCGGCACAGACCGTGACTACACGTACGACGAGCTACTAGAGCGAGTGTTCGACATCATGCGAGAAAAGAnnnnnAGCATGGTTT >ise2c.pk002.m10 SAR1 SEQ ID NO: 17GCACGAGGCAGATTCATATTTCCATCGCTTATTCGTTGCTGAGAAAAATCGTCGGTTTTAGCGACGTAACATATTGCTAATAAGTGTGAAATATTGTGATAAACTTCCTTTTAGCATTAGTTAATCTAGTTCAATTTTAAATAATTCAAAATGTTTATCTTGGATTGGTTCACTGGTGTTCTCGGATTCCTTGGTCTGTGGAAGAAATCAGGCAAGCTACTGTTCCTGGGACTGGACAATGCTGGCAAGACCACACTCCTGCACATGCTGAAGGATGACAGATTGGCGCAGCATGTACCCACATTGCATCCCACGTCGGAGGAACTGTCAATAGGCAGTATGCGTTTCACGACGTTCGACTTGGGCGGGCATCAGCAGGCGCGGCGCGTGTGGCGCGACTACTTCCCGGCGGTGGACGCCATCGTGTTCCTGGTGGACGCGTGCGACCGCCCGCGCCTGCCCGAGTCCAAGGCCGAGCTGGACTCGCTGCTCACTGACGAGACGCTCAGCnnACTGCCCCGTGCTCATCCTCGGCAACAAGATCGACAAGCCCGGCGCAGCTAGTGAGGACGAGCTCCGTCAGTTCTTCAACCTGTACCAACAGACCACTGGAnAnGnCAAAGTATCnAGnTCAnnnnT >ise2c.pk001.c14 Elongation factorSEQ ID NO: 18GCACGAGGGTCTATCTCGGATATTACACGTGGATTGTAATCCGTGACTAACCAAAAATGGGCAAGGAAAAGnnnCACATTAACATTGTCGTCATTGGACACGTCGACTCCGGCAAGTCCACCACCACCGGTCACTTGATCTACAAATGCGGTGGTATCGACAAACGTACCATCGAGAAGTTCGAGAAGGnnnCCCAGGAAATGGGGTAAGGGTTCCTTCAAATACGCCTGGGTATTGGACAAACTGAAGGCTGAGCGTGAACGTGGTATCACCATCGATATTGCTCTGTGGAAGTTCGAAACCGCTAAATACTATGTCACCATCATTGACGCTCCCGGACACAGAGATTTCATCAAGAACATGATCACTGGAACTTCCCAGGCTGATTGCGCCGTACTCATTGTCGCCGCTGGTACCGGTGAGTTCGAGGCTGGTATCTCGAAGAACGGACAGACCCGTGAGCACGCTCTGCTCGCTTTCACACTCGGTGTCAAGCAGCTGATTGTGGGCGTCAACAAAATGGACTCCACTGAGCCCCCATACAGCGAATCCCGTTTCGAGGAAATCnnnnnnn >ise2c.pk001.d16 Elongation factorSEQ ID NO: 19GCACGAGGCGGATATTACACGTGGATTGTAATCCGTGACTAACCAAAAATGGGCAAGGAAAAGnTTCACATTAACATTGTCGTCATTGGACACGTCGACTCCGGCAAGTCCACCACCACCGGTCACTTGATCTACAAATGCGGTGGTATCGAnnnACGTACCATnnnnn >ise2c.pk001.j9 myosin SEQ ID NO: 20GCACGAGGCTCTAGTCCCGTCACCGTCGCCAGTAGGGGGCGCCACAAGAACAGAAAGAGAATTATTTCAAACTCCAATTATAACCTACTAGATAACTCCAAAAGTTCTGTCAGTTCTAACTTTAATTTAACGGGGACGTCAGAGTTTATGGATAGGACCGATAAGATAATATCGGACGCGACTGAGCTACAAGCAATGCAGAACTTTATCATGGAGAAGATTTACGAAATGGAACCTAATGAGAAGAAGAAGCAATCTGAGGTCGACAGGGTATTCAAACACGCATTATTAGAATTCAAAGACAATTTAGTAGCGACGTACAGCATAGTGGAGACGCGGGGCTCTGCGCTGAAGTACAAGGATCTGATCGGCAACTTCCTGCACGTCATGGAGACGGTTTGTGCCAGGGAGGGGTCCACGCTCTCCATCACCATGGGGGTCAACGCCTTTAGGGGTTTCATGGACGAGTTTATGAGCCAACATGACACTGATAAAGCTAGGACGnnnnGnnnAAGGATAAAAAGAnnnnnnTGGACGATCCAATACAATACAAAGGCCATACGTTCATACTGTCCATGATCAACATACCAAnnnnAGTGTGAGATCTGCAAGACTTTCTTCATGTGGCCCATAGAGCGGTCACTCATATGCCAGACGTGTAAACTTGCCTCGCATAAnnnTnnnACACTA >ise2c.pk001.b14 potassium channel amino acid transporterSEQ ID NO: 21TGACTCCACAGTGGGACAAACTCATAGAGCTTGATGTGTGGTACGCTGCTGTGACCCAAGTGTTCTTCTCTCTGTCTGTGTGCACCGGTGCCATCATTATGTTCTCGTCCTACAATGGATTCAGACAAAATGTTTACAGAGACGCGATGATTGTCACTACTTTGGACACCTTCACCAGTTTGTTATCCGGTTTCACGATCTTCGGTATCCTGGGTAACTTGGCGTACGAGTTGGACAAAGATGTGGATGACGTCACTGGTTCTGCAGGAACTGGACTTGCCTTCATTTCATACCCTGACGCGATCTCCAAAACTTTCCAGCCACAGTTGTTCGCAGTGCTGTTCTTCTTGATGATGACGGTACTAGGTATCGGATCAGCAGTTGCTTTACTTTCCACCATCAACACCGTGATGATGGACGCGTTCCCTCGCATCAAGACCATCTACATGTCCGCCTTCTGCTGCACTATTGGATTTGCCATCGGTCTCATTTACGTCACACCTGGTGGCCAATATATTCTCGAGCTGGTGGATTACTTCGGTGGAACCTTCCTGATTCTCTTCTGTGCTATCGCTGAAATTATTGGTGTATTCTGGATTTACGGCTTGGAGnnnTATGCCTGGATATTGAGTACATGTTGGGAGTTAAACTTCTTCTACTGGnnnTnnTGTTGGGGCGTTATTATGCCTGCCATGATGATnACCGnnnnnn >ise2c.pk003.f2 potassium inwardly rectifier . . .SEQ ID NO: 22GCACGAGGGTAAACAGATTTTAACACTACATTAATTTGTTCTAGAGTTAAATGTATTAATTCCGACTTAAAAACAGTGCTTGTGATAAGTGAACACAAATTATTGAGCAATGACTGACTTTATAAAACAATATTTCAAGGAACAATATGAAATAAATGAAAAAATGCTTTCGAAAATTGACGCGGATCTGCGAACCTGCGGAGCACACTTAGTAGCAGTGAAGTTAATGGTGACTGCCCTCGAGTTGAAAATGACTTCGATGAAGACAATGTATCAGGATCTAATGGAACTCAGAGAAATAATCGTTCTTTTAAATCCACACTTGAAGAAACCGAGATAATAATACAATACAGTAAGGTTAACGAATACTATCTTTTAATTTCCTTAAATTATGTTCATAAAAATGATTAAGTTGTTTAGCTGAACACAGTGGTGTACTGACAGGATAGGTTTCATTAAACTTTGCATAATCGATCAGAAAACCGTGCTTTTCTTTTTTGTACTCGACCATTTCAATAAAGCGATGACCCCATAGGATTnnnnTGGGTGGTGTAGCTCGACTTCGCTTGGACAGGCTGACCAGTTGATTCTATAGTGCCTTCAAACACTACGAnnATTTCCATAT >ise2c.pk005.l20 amino acid transporterSEQ ID NO: 23GCACGAGGATTTTCTTAAAACGGTACTGCAGCAAAAAGACGGCATTGAAGGTGGACTCGGTCTGCCTATCTGGTACCTGGTGGTTTGTCTGTTCGGGTCATGGTTTATCATCTTCGTGATTGTGTCCCGAGGTGTAAAGAGTTCCGGTAAAGCTGCATACTTCTTGGCTCTCTTCCCCTACGTTGTGATGCTCATTTTGCTTATAACGACCTCTATTCTGCCCGGAGCCGGCACCGGCATTCTTTTCTTCCTGACTCCACAGTGGGACAAACTCATAGAGCTTGATGTGTGGTACGCTGCCGTGACCCAAGTGTTCTTCTCTCTGTCTGTGTGCACCGGTGCCATCATTATGTTCTCGTCCTACAATGGATTCAGACAAAATGTTTACAGAGACGCGATGATTGTCACTACTTTGGACACCTTCACCAGTTTGTTATCCGGTTTCACGATCTTnnnTATCCTnnnTAACTTG >ise2c.pk001.d1 tubulin SEQ ID NO: 24GCACGAGGCCGGTCTTCAGGGCTTCCTTATCTTCCACTCCTTCGGTGGAGGTACTGGATCTGGTTTCACTTCCCTCCTGATGGAGCGACTCTCCGTGGACTACGGCAAGAAGTCCAAGCTGGAGTTCGCCATCTACCCGGCGCCTCAGGTGTCCACCGCTGTCGTGGAGCCCTACAACTCCATCCTCACCACCCACACCACCCTTGAGCACTCCGACTGCGCCTTCATGGTCGACAACGAGGCCATCTACGACATCTGCCGCCGCAACCTCGACATCGAGCGCCCCACGTACACCAACCTGAACCGTCTCATCGGGCAGATCGTGTCCTCCATCACGGCCTCCCTGCGCTTCGACGGCGCCCTCAACGTCGATCTTACCGAGTTCCAGACCAACTTGGTGCCCTACCCCCGTATCCACTTCCCTCTGGTCACATACGCCCCGGTCATCTCTGCCGAGAAGGCGTACCACGAGCAGCTGTCGGTGGCTGAAATCACCAACGCATGCTTCGAGCCCGCCAACCAGATGGTCAAGTGCGACCCTCGTCACGGCAAGTACATGGCTnnnnTGCATGTTGTACCGTGGTGACGTCGTCCCCAAGGACGTGAACGCCGCCATCGCCACCATCAAGACCAAGCGTACCATCCAGnnnCGTCnnTTGGTGTCCnnCnnnGTnnn >ise2c.pk001.k6 tubulin SEQ ID NO: 25GCACGAGGATTCGTTTGGCAAGCCTCTTAACCGGTCGCGCTGAACGACGACTGATATTTAATTAATTTATATTCTACGTTAAGTTCAACAAAACTCAATTCAAAATGCGTGAGTGCATCTCAGTACACGTTGGACAAGCCGGAGTCCAGATCGGTAATGCCTGCTGGGAATTATATTGCCTTGAGCATGGAATCCAGCCTGACGGCCAGATGCCCACAGACAAGACCGTGGGCGGTGGTGATGACTCCTTCAACACCTTCTTCAGCGAGACCGGTGCCGGCAAGCACGTCCCCAGGGCTGTGTTTGTTGACTTGGAACCCACAGTAGTTGATGAGGTCCGCACTGGCACATACAGACAGTTGTTTCATCCAGAACAACTTATCACTGGTAAGGAAGATGCGGCCAACAACTACGCCCGTGGTCACTACACCATCGGCAAGGAAATCGTAGACCTAGTCCTCGACCGCATCCGTAAGCTCGCCGACCAGTGCACCGGTCTCCAGGGCTTCCTTATCTTCCACnnnnnTCGGTGnnnnnACTGGGATCTGGTTTCACTTCCCTCCTGATGGAGCGACTCTCCGTGGACTACGGCAAGAAGTnnAAGCTGGAGTTCGCCATCTnnnCnGCnnCTCnnnnnTCnnnnnnCTGTC >ise2c.pk001.l2 tubulinSEQ ID NO: 26TTCGGCACGAGGGGCAAGCCTCTTAACCGGTCGCGCTGAACGACGACTGATATTTAATTAATTTATATTCTACGTTAAGTTCAACAAAACTCAATTCAAAATGCGTGAGTGCATCTCAGTACACGTTGGACAAGCCGGAGTCCAGATCGGTAATGCCTGCTGGGAATTATATTGCCTTGAGCATGGAATCCAGCCTGATGGCCAGATGCCCACAGACAAGACCGTGGGCGGTGGTGATGACTCCTTCAACACCTTCTTCAGCGAGACCGGTGCCGGCAAGCACGTCCCCAGGGCTGTGTTTGTTGACTTGGAACCCACAGTAGTTGATGAGGTCCGCACTGGCACATACAGACAGTTGTTTCATCCAGAACAACTTATCACTGGTAAGGAAGATGCGGCCAACAACTACGCCCGTGGTCACTACACCATCGGCAAGGAAATCGTAGACCTAGTCCTCGACCGCATCCGTAAGCTCGCCGACCAGTGCACCGGTCTCCAGGGCTTCCTTATCTTCCACTCCnnnCnGTGGAGnTnnnTGGATCTGGTTTCACTTCCCTCCTGATGGAGCGACTCTCCGTGGACTACGGCAAGAAGTnnAAGCTGGAGTTCGCCATCTAnnnn >ise2c.pk002.b4 ubiquitin SEQ ID NO: 27GCACGAGGATCAAAGAGTTACGAACCGTCACCATACTGAAGGAGATACCATTCGTCGTGCCATTCTCAACACGCGTCCTTATATTCCAAGGACTTTTAGCGAGAGAGAAGCACGACCACTGGTACGAAATGACGAACTTCAACGAGGGGCCCTCGATCAACATCAGTGTTCGAAGGACGCATTTATATGAAGATGCATTTGATAAACTTAGTCCGGATAATGAACCTGATTTGAAGTTGAAACTTCGCGTGCAACTGATCAACCAGGCCGGTGCGGAGGAAGCTGGTGTCGACGGCGGTGGACTATTCCGAGAGTTTCTTTCTGAGCTCTTAAAATCTGCATTTGATCCGAACAGGGGTCTGTTCCGGCTGACAATAGACAACATGTTGTATCCGAACCCCGCCGTACATCTACTGTACGATGACTTCCCCATGCACTACTACTTCGTCGGCAGGATGCTGGGAAAGGCGATGTACGAGAACCTGTTGGTGGAGCTGCCGCTGGCGGAGTTCTTCCTGGGCAAGCTGTGCGGCTGCGGGGAGGCCGACGTGCACGCGCTGGCCTCGCTCGACCCCGCGCTGCACCGCGGGTTGTTACTACTC >ise2c.pk001.j16 small nuclear ribonucleoprotein SEQ ID NO: 28GCACGAGGGCCGGCCGCCGTGTTCGTGCCGTCCCGCCGGGCCGCGCGCCTACTGGCCGCCGACCTGCTGGCGCTGGCCGCGGCGCACGCGCAGCCCGCCGCCTTCCTGCGCGCGCGCCCCGACGTGCTGCAGCCCTTCCTCAAGAGGATCAACGACAAGATGCTGAAGGAGACGGTGGCTGCGGGCGTGGCGTACCTGCACGAGGGCGTGGACCCGGCGGAnnGGCGCCTGGTGCAACAACTGCTGGAGTCGGGCGCGCTGGCGCTCTGCGTCGTGGCCGCCGAGCTGGCCTGGGGACT >ise2c.pk006.h23 small nuclear ribonucleoproteinSEQ ID NO: 29GCACGAGGCGAAGATAAAGGTCGCGTGTGGACCTTAGGTTTAAGTTTATTATTAAATAATTTAGCCTAAACATAAGTCATGGCCAATAACGACAACTTTGCACAAGATGTTACTGATAATCAACTAAATGGAAATGCCGAAAATGGTGGTGGCGATACGCAAGAACATAATAGTGCCGAAGCCCCTGGGCGTGATGATGACAGAAAACTTTTTGTCGGAGGCCTGAGCTGGGAAACCACAGACAAGGAGTTACGTGACCACTTCAGTGCATATGGTGAGATTGAGAGCATCAATGTCAAGACTGATCCAAACACTGGCAGATCAAGAGGATTTGCCTTTATTGTGTTCAAGGCACCAGATTCAATAGACAAAGTGATGGCTGCTGGAGAGCACACTATTAACAACAAAAAAGTTGATCCGAAAAAAGCAAAGGCTAGACATGGAAAGATCTTTGTTGGTGGTCTTAGCAGTGAAATATCAGATGATGAGATCAAAAACTTCTTCAGTAATTTTGGAACAATAATTGAAGTCGAGATGCCCTTTGACAAAACCAAGAATCAGnnnAAGGGATTCTGCTTTATAACATTCGAGTCTGAACAGGTGGTCAATGAGCTGCTGAnnnCn >ise2c.pk006.m8 SEQ ID NO: 30GCACGAGGCGCGTGTGGACCTTAGGTTTAAGTTTATTATTAAATAATTTAGCCTAAACATAAGTCATGGCCAATAACGACAACTTTGCACAAGATGTTACTGATAATCAACTAAATGGAAATGCCGAAAATGGTGGTGGCGATACGCAAGAACATAATAGTGCCGAAGCCCCTGGGCGTGATGATGACAGAAAACTTTTTGTCGGAGGCCTGAGCTGGGAAACCACAGACAAGGAGTTACGTGACCACTTCAGTGCATATGGTGAGATTGAGAGCATCAATGTCAAGACTGATCCAAACACTGGCAGATCAAGAGGATTTGCCTTTATTGTGTTCAAGGCACCAGATTCAATAGACAAAGTGATGGCTGCTGGAGAGCACACTATTAACAACAAAAAAGTTGATCCGAAAAAAGCAAAGGCTAGACATGGAAAGATCTTTGTTGGTGGTCTTAGCAGTGAAATATCAGATGATGAGATCAAAAACTTCTTCAGTAATTTTGGAACAATAATTGAAGTCGAGATGCCCTTTGACAAAACTAAGAATCAGAGGAAGGGATTCTGCTTTATAACATTCGAGTCTGAACAGGTGGTCAATGAGCTGCTGAnGACTCCTAAGCAGnnnATTGGTGGCAnnnnnnnCGAC >ise2c.pk001.a23SEQ ID NO: 31GCACGAGGATGAAGTTGGCTCTGACACTCTTGGCTCTGGCGGCGGTGGCCACCGCTAAAAACATCAACGTCGAGGATGCCATCGACCTAGAGGACATCACCGCCTACGGATACTTGGCTAAGATCGGTAAACCTCTTGCCGACGAAATCCGCAAAGCTGAGGAGGCAGAGAGCGCATCCAGAATTGTTGGTGGTCAGGCCTCCAGCCTCGGACAGTTCCCCTACCAGGCTGGTCTTCTCGCTGACTTCTCCGCTGGCCAAGGTGTGTGTGGTGGTTCCTTGGTGCGTGCCAACCGTGTTCTTACTGCTGCTCACTGCTGGTTCGATGGCCAGAACCAGGCCTGGAGATTCACCGTTGTTCTTGGCTCCATCCGTTTGTTCTCCGGTGGTACCAGAGTTCAAACCTCCAACGTTGTTATGCATGGAAGCTGGAACCCCAGTAACATCCGTAATGACGTCGCCATGATCAGGCTGAACTCCAACGTTGGTCTTTCAAACACCATTGCACTCATCGCTCTGCCCAGCGGTAGCCAGCTCAACGAAAACTTCGCCGGTGAAAACGCCGTCGCnnnCTGGATTCG >ise2c.pk001.a7SEQ ID NO: 32GCACGAGGATCAAAATGAAACTGTTCCTCGCAGTCGTGTGCTTGGCCGTTGCCGCATCCGCGGTGGAGATTGGAGTTCCGTCTCAGGAAAACCCAGTCTTTGGCTACCATCAAAACTTCGGTATTGCCGAAGCTGCCAGGATCAAGAAGGCTGAGGAAGAAACCAGCCCTAGCGCCCAGAGGATCGTCGGAGGATCTGTCACTGACATTTCCAACGTCCCTTACCAGGCTGGTCTCGTGATCCAAGTTTTGGTCATCTTCCAATCCGTGTGCGGTGGTTCCATCATCTCCCACAACCGCATCGTGACCGCTGCTCACTGCAACTGGGACGGTTCTATCACCGCTAACTCTTTCACCGTCGTACTTGGCTCCAACTTCCTCTTCTCCGGCGGTAACCGCATCACCACCAGAGATGTTGTCATGCACCCCAACTGGACCCCAACCACCGCTGCCAACGACATTGCTGTCCTCCGCATTAGCTCCGTTACTTTCACCAACGTGATCCAGCCCATCGCTCTGCCCAGCGGCAACGAGCTCAACAACGACTTCGTCAACTGGAACGCTATCGCTTCCGGATACGGTCTTACCGCTGATGGTGCTAACATCGGTACTACCCAACGTGTCAGCTCCGTGGTACTCCCCGTGATCnnnnnnCGCCAGnnCGCTACCGTnnnnnn >ise2c.pk004.c4 SEQ ID NO: 33GCACGAGGAATCTTAGTTACATTGGAGTGACTTTTATTTATCAATAACATTTTTATTTGAAGACTCAGTACGTATTATCGCGTAGTTCAACAGAGTTGCTAGTGTAGTTTTCTGAAAGTTGCCATCTTGCTTTTGCAACTTTTAAATATAAAAGTCTTATTAGATCGTTTTTACTACCGATAAATTTACTAAAAATATAAAAGTGCAATTTACAATTACTCTGTTAGTGTCAGTTTGTGTGAATTTGTCGTAGTTATAAAAGGACACTGTATTGATTTTGTCAATCAGTTTGACGCATGCGCTCATTGGGTGCCGTAAAAAAGGGTTGGCCAACATTCCGAACAGTGTCGTTCCGGTCGCCGTTGTCGTGGTGTCGGTGAAGTTAGTGGTGGAATTTTTACGTGTATAACATCAAAAAATGGCGTCTGGTGTGACAGTTTCGGACGCGTGCAAAACGACGTACGAGGAGATTAAGAAAGACAAGAAGCACCGCTACGTGGTGTTCTACATCAGGGATGAGAAACAAATTGACGTAGAGACCGTCGGCGAACGTAACGCGGAATACGATCAGTTCCTTGAGGATCTGCAGnnnGGTGGCACCGGnnAGTGCn >ise2c.pk004.l4 SEQ ID NO: 34GCACGAGGCTGATATCTAATCTTAGTTACATTGGATTGACTTTTATTTATCAATAACATTTTTATTTGAAGACTCAGTACGTATTATCGCGTAGTTCAACGGAGTTGCTAGTGTAGTTTTCTGAAAGTTGCCATCTTGCTTTTGCAACTTTTAAATATAAAAGTCTTATTAGATCGTTTTTACTACCGATAAATTTATCAAAAATATAAAAGTGCAATTTACAATTACTCTGTTAGTGTCAGTTTGTGTGAATTTGTCTTAGTTATAAAAGGACACTGTATTGATTTTGTCAATCAGTTTGACGCATGCGCTCATTGGGTGCCGTAAAAAAGGGTTGGCCAACATTCCGAACAGTGTCGTTCCGGTCGCCGTTGTCGTGGTGTCGGTGAAGTTAGTGGTGGAATTTTTACGTGTATAACATCAAAAAATGGCGTCTGGTGTGACAGTTTCGGACGCGTGCAAAACGACGTACGAGGAGATTAAGAAAGACAAGAAGnnnCCGCTACGTGGTGTTCTACATCAGGGATGAGAAACAAATTGACGTAGAGACCGTCGGCGAACGTAACGCGGAATACGATCAGTTCCTTGAGGATCTGCAGAAGGGTGGCACCGGAGAGTGCAGATATGGCCTCTTCGACTTCGAGTACACGCACCAGTGCCAAGGCACGTCGnnn >ise2c.pk004.n19SEQ ID NO: 35GCACGAGGCCTCGTGCCGCGCGAATAGACAGTTTTGTGTGCACAATGTTGATCCTTTGGCTAAATATCATCGCAATAATTTGTGTCATACCCTACGCAAATGGAGAAGGAAGGGTTGCAATAGCGCATTTACAATCGCTAAAGTCAGTGACTGGTCAAATTCAATTTACGGAGACGGCAAAAGGGCTTCATGTCGAAGGAGTTATATTTGGTTTACCACCCGGTGCCTACGGGTTTCACGTTCACGAATTAGGAGATGTTGCACCTGGTTGCGACCAGGCGGGCCGGCACTTCAACCCTGAGGGATCCACCCACGGTGGCAGGAACTCCACCGTACGCCATGTCGGTGACCTCGGAAATGTAGTGTTCGTTAGCGAGCGAGCCGCTTATGCTACAGTAGACTTTGTAGATAGTCTATTGGCACTTCAAGGACGTAATAGTATATTGGGGCGCTCTTTGGTCTTGCATGAACAAACGGATGACCTAGGTTTGGGAGGAAACGCGACGTCTTTGACTACAGGTAACTCGGGGCCCCGGATAGCATGTGGTGCTATTGGAATCAAATCACCTTATGACCCTTGGAATGCTGCTAGCTCTATGTCTCCGTCGATGCTACTATTTATCACATCTTTAACTTTATTTACTTTAnnnTnnnAAnTnnnnGTATnAGTATTTAATTTnnnnn >ise2c.pk005.f21 SEQ ID NO: 36GCACGAGGCTTCCACATACGCGAATAGACAGTTTTGTGTGCACAATGTTGGTCCTTTGGCTAAATATCATCGCAATAATTTGTGTCATACCCTACGCAAATGGAGAAGGAAGGGTTGCAATAGCGCATTTACAATCGCTAAAGTCAGTGACTGGTCAAATTCAATTTACGGAGACGGCAAAAGGGCTTCATGTCGAAGGAGTTATATTTGGTTTACCACCCGGTGCCTACGGGTTTCATGTTCACGAATTAGGAGATGTTGCACCTGGTTGCGACCAGGCGGGCCGGCACTTCAACCCTGAGGGATCCAACCACGGTGGCnnnnnCTCCACCGTGCGCCATGTCGGTGACCTCnnAAATGTAGTGnTTGTTAGCGAGCGAGCCGCTTATGCTACAGTnnnCn >ise2c.pk010.h5 SEQ ID NO: 37GCACGAGGGTCGAGAGATACGGTGCGCACATAGCAACAATATCAAAGTACAAAGGTCAGTAACTATGAGTGGTAAATTGTTAAAAACTCTAATCCTTGGGGCACCTGCTTCAGGCAAGGGGACTATATCGTCTCGGATAGTGAAGAAATATGCTGTGGCACACGTGTCCAGTGGGGACAAGCTGAGGGACCACATTGAGAAACAAACTGACCTAGGTAAAGAAGTCAAAAAGTACTTGAATGAAGGGAAACTTGTACCTGATGATGTCATGATAAAGTTTATGATCACAGAATTAAAAAAAGTTGAAGATAAACCATGGCTACTGGATGGATTCCCGAGGACTGTGGGACAGGCTGATGCTTTGTGGAAGGTACAACCTGTTGATGTAGTAGnnnnnTTAGTAGTGCCTTTTGAGGTAATCATAGACAGAGTGAnnnAnCGCTGGGTGCACTTGCCTTCGGGCCGAGTGTATAACATTGGCTTCAACACTCCTAAAGTGGAAGGTAAGGATGATGAGACAGGTGAGGACTTGGTTCAGAGACCTGACGACAAGCCAGAGGCTGTGCGCAAGCGGCTGGAGATCTATGAGAGTGTGACGAGGCCAGTCATAGAGTTCTAnnnnGCTAA >ise2c.pk001.c18 SEQ ID NO: 38TGCTGCTGCTGGAAGCTGGGCCCAACCCTCCCGAGGAGAGCATTATACCAGGCTTAAGACAAACCTTGAAAGAAACGCCCTACGACTGGAACTTCACCACCATTGACGACGGGGTCACGAGCCAGGCGCTGGCGGGCCACGTGCAGAGACAGCCGCGGGGCAAGATGCTGGGCGGCAGCGGCTCGCTCAACGACATGGTGTACGCGCGGGGCCACCCCGAGGACTACTACGAGTGGGCCGACATCGCCGGCGACGTCTGGAACTGGACCAACGTGCTGGACTACTTCAAGCGGACGGAGCACATGACGGACGCCAATATCGTTCACAACnnnnAGCTCATGCAGTACCACGGCACGGnnnnnnCCATnnnnnnnTnnnGnnnCCAnTnnnnnnnn >ise2c.pk004.p1 SEQ ID NO: 39GCACGAGGGGAAAACATGGGAAGGAGGTCGCATCAAGATGTTAGTGCTCGACTTGAACTGCCCGGTCGTTGGAGACGACTGCAAAGACAGCCGCAAGAAGTTGCTTGTGGACTACTTCCATACAAACCTGCATACCCAGAACTTCTACGCGTTCCGCTTCTTTATCTGCGAAGTGTTGAACTTCATCAACGTCGTGGGCCAGATCTTCTTCATGGACTTTTTCCTGGACGGCGAGTTCTCCACGTACGGCAGTGACGTGGTCAGTTTCACCGAGATGGAGCCCGAGGAGCGTGTGGACCCGATGGCTAGAGTGTTCCCGAAAGTGACCAAGTGCACCTTCCACAAATACGGTCCTTCAGGAACCGTGCAGAAGTTCGACGGTCTGTGCGTGCTGCCATTGAACATCGTCAATGAAAAGATCTACGTGTTCCTGTGGTTCTGGTTTATGATCCTGTCGATCCTGAGTGGAATTTCGCTGATTTACCGCATGGCCGTGGTGGCTGGACCGCGCGTGCGCCTGTACCTGCTGCGTGCGCGCAGCCGCCTGGCCCCGCnnnCGCnnnnnGnnnn >ise2c.pk005.p13SEQ ID NO: 40GCACGAGGATTTTAATAGCTATTATGACTTTACAGACTAGACGGATCAAGGCCATGCCTCTCGCTTGCATACTCACCATCCGCACATACCGTATTGCGGTATGTCAATAAGTTGCAAATAATGTCTGTTCAGTTTTACAAGGATAAGATCAGCAGTATTTGCGAACTGTACCTACTACTAAGCTGATAATGTAATAATTAAACTTTATTATTGAAATAGATATGTATAATTGACATCTTTCTCAAATGGGTGTCAATACTGCCAACTCTATTACCACAATTTCTTTTCGTATTTGCTTTTATACTGAGCCTGATGACGTACTGTACTTTTTATTAGAATTTAATTTTTCTTATTTTTCTTACTACGTAGTCATTAAATCTGAGAAATTAAAAATTACTAATTTAGAACTCCCAAATTCTGAATGAGGTTCTAAAAAGTTGTTAGGAATACTAAATACCATTTTACCAACATAAATCTAATTTCGTTACTTAAAATATTAAATGTATAATGAAATGTCTATGATAAGTGTTTACTATCTTTATATCGACAAAATTTATTTTCCATGTTTTAAAATTTATTTTTCAGATGTTTTGACGTGATAAGTTTGTATTTTATCAATATCTGATAGTCGAGAGTTAnnnAnTATTG >ise2c.pk001.f12SEQ ID NO: 41GCACGAGGGAGGAGAGGTGGTGGCTGGCTTCCTTGCAAACGAAGCGTCGTAAATTACATCTTATTTGTAAATTTTAATAAAAATTTGATCGTTAAACGATCGAATCAGTAGTGATTTAAGTGCTCAAGCAGTTTCACATCCAATCGACAATGAGTTCGAGTGTATGCTACAAGTGTAACCGGACAGGGCACTTCGCCCGCGAGTGCACCCAGGGTGGTGTTGCCGCTCGTGACTCTGGTTTCAACCGTCAGCGCGAAAAGTGCTTCAAGTGCAACCGCGCTGGGCACTTCGCTCGGGATTGCAAGGAGGAGGCCGACCGTTGCTACAGATGTAACGGCACGGGACACATAGCGCGTGAGTGCGCGCAAAGTCCGGACGAGCCGTCGTGTTACACTTGCAACAAGACCGGGCACATCGCACGGAACTGCCCAGAGGGCGGGCGCGACAGCTCCAACCAGACCTGCTACAACTGCAACAAGTCCGGCCACATCTCACGCAACTGCCCCGACGGCACCAAGACTTGTTACGTGTGCGGAAAGCCCGGACACATCTCCCGCGATTGCGATGAGGAGCGGAACTAACACACGCCTCTTCGCGACTGCCTATATATAnnnTAAACTATGTATATTATGATGCCACGCACGGACGATAAGCAAAGGACGCGATACGCGACACTAGATCGTAAGACCACACGACTGTATGnnnnTAATGCAACG >ise2c.pk001.n21 SEQ ID NO: 42GCACGAGGATAATAAACGTTAATATTTAACAAGTTGAAAAGTTTGTCTTTCAATTTGTGATTTTGTAAAGATCATTCTATGGAATGGACAGTTTGCTATCTGTGAAACATCCATTAGCTTTGTGTTGAGAGCAGAGGTCGCGGCGGCGGGGTGATGCGGCCATGGCTTCGCGGCGCGTGACGCGCAAGTGGGAGGTGTTCGCGGGACGGAACCGATTCTGGTGCGACGGCCGCCTCATGACGGCGCCGCACCCCGGCGTGTTCCTGCTCACGCTCGCGCTCATCTGCGGCACGTGCGCCCTGCACTTCGCCTTCGACTGCCCCTTCCTGGCCGTGCGCGTGTCGCCCGCCGTGCCCGCGGCCGGCGCCGCGCTGTGCGCGCTGACGCTGGCGGCGCTGCTGCGCACGGCGCTGTCCGACCCCGGCATCATCCCGCGCGCCGCCGCGGCCGAGGCGGCGGCGCTGGAGGCGGnG >ise2c.pk004.e20 SEQ ID NO: 43CGCGCACGTCGCTCnnCAAGCCCGCTGCAGCGCCGGCCAAGCCCGCCCCCGCGGCGGCGCGCGCCACCAGTGCGACCAGCCGCGCGGCCCCCGCGGCCCGGCCGGCCCCCAAGTCCGCAGTAGGCGCAGCGCGGCCCGCAGCACAAAAGACAGATGCGGCCGCCAAACCCGCGGCGACCCGGGTTGCGGCTCCGCGTCCCGCGCTGTCGGCGCCCAGGCCCCAGCCTAAGCCGGCAGACAAGAAGCCAGTACCGAATGGTGACGTGAAAGACTCCAAGCCAGCCGCGCGGCCCGCGCCCCGGCCGGCCGCGGCCGCGCGCCCCGCGCCGCGCCCCACTCCCCGCGCCCCCGCCGCACnGGTCGCACCCACTACTnnnnnGAGTGCCCCCAAGCCGGCGCCGCGTGCTCCCCTGGACAAGCAGAGCnnnGACCTCGCTAACAAACGCATCnnnGnCAnGGCAGCACCGCCTAGGACTGCTCCCCCTAAGACGACAACGACGACAACAGGnnnnnnnnnnnnnGTnnCGAAGnnnnn >ise2c.pk005.n11 SEQ ID NO: 44GCACGAGGCTATAACAAGCAGCATATAAAAATGAAATTCTTGCTGTCTTTCGCTGCCGTCATCGCCGTGGCCGCCGCTGGCCTGGTGCCCGTTGGACCCGCCGGCCCTGCGCCCGCTCCTGAGGCCCCTGAGGTCTTCGAGCCCGTCGCTATTGGACCCGCTGTCATTGACTCCTTCGAGCCCATCGCCATCGGACCCGCTATCATCGACTCCTTCGAGCCCATCGCCATCGGACCCGCTATTGTTCCATCTCCCGAGCCCGTCGCCATCGGACCCGCCATCATTGAGAGCCCAGAGCCCGTTGCTGTCGGACCTGCATGGATTGACTTCCCCCTGCCCGACGGTGGTGCTGCCGTTGCCCCCGTTGAGCCCTCTCCCGTGGCTGTTATCCCCGGTCCCGTGTCCACTGAGGTTGCTTCAGGCACTCCCCTCGTTCAGATCATCCTGAACATCAACnnnnnnTCTGCTGACGTTAGCCCCGTTGCTGTnGGCCCCGCTGTCGAGnnnACACCCGTGCACGTTGTGGACTCTGCCCCTGAACCCGTCCACGTTGTGnnnnnnGCCCCnnnnnCnATCnnnnnGTCGn >ise2c.pk003.l14SEQ ID NO: 45GCACGAGGCTTAGAGTAAGCATAGGTGTATTTATGTATTGAGTCGGAAGAAGCAATGGACGATCCAAATAGGATGATGGCGCATAGCGGCGGGCTTATGGGGCCGCAGGGCTACGGCCTGCCTGGCGGCGAGGGAACTCCAACCGCAGGCGAAGGTGAAGCCCGCAAGCAAGATATTGGTGAAATATTGCAACAGATCATGAATATTACAGATCAAAGTCTTGATGAAGCGCAAGCGAGAAAACATACTCTCAACTGTCACAGAATGAAGCCTGCCCTATTTTCAGTGTTGTGTGAAATCAAAGAGAAAACAGTGCTGTCCCTCCGCAACACGCAAGAGGAGGAGCCCCCAGATCCCCAGCTGATGCGCTTGGACAACATGCTCATAGCCGAGGGGGTCGCTGGCCCTGAAAAGGGTGGTGGTGCGGGCGCTGCAGCTTCGGCATCAGCTGCTGCTGGTGAATGGGACAATGCCATCGAGCACTCTGACTACCGTGCGAAGTTGGCGCAGATCCGCCAGATCTACCACCAGGAGCTGGACAAGTATGAGAATGCTTGTAATGnnnnnnCCACCCACGTGATGAACTTACTCCGCGAGCAGAGCCGCACCAGGCCTATCACAnn >ise2c.pk003.e24 SEQ ID NO: 46GCACGAGGCCAGGTTTGAGAAAAACGCTTAAACTGCCACAAAATCCCGTTCTCGAAGAAGCACTTTTCACTTATTAATAAGTAACTTGTGTAAAATGTGGTTTAAATGTGTATTTTACTAAACCTCAATAAATATATTTATATCAAAATATTTTTTTTCTATACTGTATTATTTATTCCTATAGTACATATTATAATCCGAACGCTCCGTGAGTCCGAACAGGGGTAATTTTTTGGTAGTTCGGATTATCGAGGCTCTACTGTATACCTACTTTTTGTTAAAATATTTTAGTCTTATATACGACTTCCTAACTAATCCATATCTCTTAGAGCTTTCGAATATCCATTTGCCTTTTTCTTAAAAAGATTAATAACTATTTATATATATCCCAAATATATAAAAACAACCACTCCAATTATTATTATTCAAATATGACAAACTAGATAGAATGTCCCAAGAAATTTGCAAAAAAGTAATGTTCAAATTATTAACCGAAGAACGAATTnnnGAGTGTATAATATTATACAGACATTTAGAAATTTTTAATAGGCTCCAATCGCATGAGAGGTCGCTTTAAAATTCGGCATTGGTGTGTGCGTTGCAATTTAATCTTTAACACCCnnn >ise2c.pk005.l5 SEQ ID NO: 47GCACGAGGGGACGTGTTTACAATTTACTTTCGTGCTCGTGTGATTTTAATTAAAACAGTGCTAAGTGCTCTAGGACGCTGAATAACTGATATTTGTTTTAAAAGTTGATATAAATTAATCACAATGAATAGAGATAAACGAGAACCAGAGTATCCAACGGAGTTGGAGTCTCAATTCGTAATGCGTTTACCTGAGGAGCCTGCAAAAGTTTTGAGAGAAGTGTTGAAATCCGGAGAGAACCTGAAAAACAGACTGACGATACAAATAGAAAACGACATGCGCACGGGCGAGGTAAGGTTTGATCACTGGTTGATGCACGCCAAGATCGTGGATCTACCAACCATCATAGAATCTCTAAAAACGATCGACAACAAGAGTTTCTACAAAACAGCAGATATATGCCAAATGATGATTTGTAAAGAAGAACCTGACCAACCATCCACAGAGGAAGAGTCACCAGCTAAAAATAAGAAAAAAGATCCATACAAAGTTGACAAAAAGTTCCTATGGCCACACGGCATCACACCGCCTACGAAGAACGTACGGAAGCGTCGATTTAGAAAAACCCTTAAAAAGAAATATGTAGAAGCACCAGAAATTGAAAAGGAAGTGAAGAGGCTGCTGAGnGCAnnCnATGAGGCTGTTAGTGTTAACTGGGAGGTCATCAAnnnnnnnGAT >ise2c.pk006.k12 SEQ ID NO: 48GCACGAGGGTCGAATGGAACATGGCGGTGCTAGGCAGGATGTGCATAAGTTTTTGATTTTTGCATTTTTAACGAGTTGCTTATATCAGTTAGCTTTCTAAATAATTTCTGACTTATTTCGTGTGTTATAATATTTGTTATAGTGTAAAAGCTTATCCACCCCAGGAATTTCCTATCTGGACTTACTTAGTTCTGCAATGAAAATTATTATTCGTTGGTAGTGTAAAAATAATTGTGACAAATATATCACTTTGCTTCAGTGTGCCGTGTTGGTCATGGCTACGCTCCTCCAAGAGAATGGTATAAAGGAGTTAAGCAAAGTTGTGCCTAACCGTGGTATATCCTCACATAGTGTAACAAATCATATGGTGCCTGATCATGAATATTGCGAAGCTGGGTCAACTAGCACGTCACAGATGAAGTGTACCGATACAAGTGAGGCGATGGCGCCACCCGCCGCCATTGAAGAAGAGGAGGATACACCAGAAATAGATATAATGATAAACAATGTTGTGTGCAGTTTTAGTGTTAAGTGCCACCTGAACCTTAGACAGATAGCATTnnnTGGTGTGAACGTTGAATTTCGCCGCGAGAACGGCATGGTAACTATGAAGTTACGGCGTCCATACACTACTGCGTCCATCTGGTCGTCCGGCCGCGTGACGTGCACTGGTGCAACCAGCG >ise2c.pk010.i8 SEQ ID NO: 49GCACGAGGGAATATTGCGAAGCTGGGTCAACTAGCACGTCACAGATGAAGTGTACCGATACAAGTGAGGCGATGGCGCCACCCGCCGCCATTGAAGAAGAGGAGGATACACCAGAAATAGATATAATGATAAACAATGTTGTGTGCAGTTTTAGTGTTAAGTGCCACCTGAACCTTAGACAGATAGCATTAAATGGTGTGAACGTTGAATTTCGCCGCGAGAACGGCATGGTAACTATGAAGTTACGGCGTCCATACACTACTGCGTCCATCTGGTCGTCCGGCCGCGTGACGTGCACTGGTGCAACCAGCGAGGACCAGGCGAAGGTTGCCGCACGACGGTATGCGCGCGCCCTTCAGAAGCTCGGCTTCCAAGTGCGTTTCCGCAATTTCCGTGTAGTCAATGTATTAGGCACCTGTCGGATGCCGTTTGGTATAAGGATCATATCTTTTTCGAAAAAATACAAGGAAGCAGACTATGAACCTGAGCTCCATCCTGGAGTCACATATAAGTTATACAATCCTAAAGCCACACTCAAGATATTCTCCACTGGTGGTGTGACTATCACAGCTCGGAGTGTGAGTGACGTTCAGTCAGCCGTGGAACGCATCTTCCCTTTGnTGTACGAGTTCCGCAAGCCTCnnnnACCGGCAnnnnA >ise2c.pk010.b12SEQ ID NO: 50GCACGAGGGTACCAAAAGCTCTTTTCATTGCAGCTGAAGGGTCACTGCAACTTGGCCAATCAGAATTAGCATTGAAACTATTCAAAGAACTAAAACAAGAAGGAATGGAAATCAGGCAACATTTCTATTGGCCTTTGTTAGTTCAGAAGGCAAAGGAAAATGATGAGGAAGGCCTCTTGCAAATTTTAAAAGAAATGAGCAGCAATGACTTTACTGTTACTGGAGAAGCGTTAAGAGACTATGTTATCCCTTACTTGATAAAAAAAGATTCTCCACAGAATGTCTTACTTAAACTTCAAATTGCAAATGTACCAACAATCCATGCTGCAAGAAATCTAATGGTTGATCTTTTGGATTCTGGAGACATAAAAGGCGCAGCGGAAATAGCTCTGCAATATAGACCTTGGGGCAACTACTCTCTTGTTGCCAGGTCCCTCATCAATGCAGTGAATAAGACAAAAGATGTAGAATCGTTTGCTAAAATTCTTCATGCTATAAGCAGTAAACCTTTGTCACAGGGTGAAGAAGATGTTGCTGCCAACAATGAGGAAGGTCAAAGTGATGAAAATAATGATATTCATGAAGTCGGCCGTATTGTGAGGTCGTCTGCCAAGAGTTTGGCTAAACCAGACTTAATAGnAAnnnnTTTAGA

TABLE 2 List of dsRNA primers.  SEQ ID NO Primer Sense AntisenseTarget/sense/ # Target Gene ID Seq ID Target strand strand antisense0075 juvenile ise1c.pk002.m13 AACATGGTATCC CAUGGUAUCC CCUGAAGUCG51/52/53 hormone diol GACTTCAGGAA GACUUCAGG GAUACCAUG kinase 0076juvenile ise1c.pk002.m13 AAGGTCGCTGAC GGUCGCUGAC CUUGUUCUCG 54/55/56hormone diol GAGAACAAGGA GAGAACAAG UCAGCGACC kinase 0077 juvenileise1c.pk002.m13 AAGTGTCCTGGG GUGUCCUGGG GAACUCAAGC 57/58/59 hormone diolCTTGAGTTCCA CUUGAGUUC CCAGGACAC kinase 0078 juvenile ise1c.pk003.f7 AAGAAGAAGCTC GAAGAAGCUC CACGUGGAGG 60/61/62 hormone diol CTCCACGTGTTCUCCACGUG AGCUUCUUC kinase 0079 juvenile ise1c.pk003.f7  AAGGTCGCTGACGGUCGCUGAC CUUGUUCUCG 63/64/65 hormone diol GAGAACAAGGA GAGAACAAGUCAGCGACC kinase 0080 juvenile ise1c.pk003.f7  AATGTCCTGGGG UGUCCUGGGGGAAACUCAGC 66/67/68 hormone diol CTGAGTTTCAA CUGAGUUUC CCCAGGACA kinase0081 juvenile ise1c.pk005.a15 AAGAATAAGCTC GAAUAAGCUC CACGUGGAGG69/70/71 hormone diol CTCCACGTGTT CUCCACGUG AGCUUAUUC kinase 0082juvenile ise1c.pk005.a15 AATTTGTCGAGG UUUGUCGAGG AUAGGGUCUC 72/73/74hormone diol AGACCCTATTG AGACCCUAU CUCGACAAA kinase 0083 juvenileise1c.pk005.a15 AAGTTCGCGTTC GUUCGCGUUC UUCAAGAGUG 75/76/77 hormone diolACTCTTGAAGA ACUCUUGAA AACGCGAAC kinase 0084 ribosomal ise1c.pk006.d24AACTGCCCCTTA CUGCCCCUUA AGAUGAGGUU 78/79/80 protein L18a ACCTCATCTATACCUCAUCU AAGGGGCAG 0085 ribosomal ise1c.pk006.d24 AATCACGCTGAAUCACGCUGAA UACAGUGGUU 81/82/83 protein L18a ACCACTGTATA ACCACUGUAUCAGCGUGA 0086 epoxide ise2c.pk009.i4  AAAATATGGCGC AAUAUGGCGCACAAUAGGCG 84/85/86 hydrolase GCCTATTGTTT GCCUAUUGU CGCCAUAUU 0087epoxide ise2c.pk009.i4  AACGTTCTCGGT CGUUCUCGGU CAGUGAAAGA 87/88/89hydrolase CTTTCACTGCT CUUUCACUG CCGAGAACG 0088 epoxide ise2c.pk009.i4 AAGTCATCGTTC GUCAUCGUUC GUAGACUUGG 90/91/92 hydrolase CAAGTCTACCTCAAGUCUAC AACGAUGAC 0089 V-ATPase A ise2c.pk001.d19 AACCCCTTGAATCCCCUUGAAU GACCUUAACA 93/94/95 subunit GTTAAGGTCGG GUUAAGGUC UUCAAGGGG0090 V-ATPase A ise2c.pk001.d19 AAGTACACCATG GUACACCAUG UACUUGCAAC96/97/98 subunit TTGCAAGTATG UUGCAAGUA AUGGUGUAC 0091 V-ATPase Aise2c.pk001.d19 AACGTGTCCATG CGUGUCCAUG GUCAGCCAUC  99/100/101 subunitATGGCTGACTC AUGGCUGAC AUGGACACG 0092 H+-ATPase V- ise2c.pk001.e14AAACCTACAAAA ACCUACAAAA UUUCGGCCAU 102/103/104 type subunit TGGCCGAAAACUGGCCGAAA UUUGUAGGU 0093 H+-ATPase V- ise2c.pk001.e14 AATCTACGGACCUCUACGGACC CCAAAGAAGG 105/106/107 type subunit CTTCTTTGGAG CUUCUUUGGGUCCGUAGA 0094 V-ATPase A ise2c.pk001.f20 AACTCTGACGTC CUCUGACGUCGUAGAUGAUG 108/109/110 subunit ATCATCTACGT AUCAUCUAC ACGUCAGAG 0095V-ATPase A ise2c.pk001.f20 AAGTGCTTGGGT GUGCUUGGGU GUCGGGGUUA111/112/113 subunit AACCCCGACAG AACCCCGAC CCCAAGCAC 0096 V-ATPase Aise2c.pk001.f20 AACTGGCTCATC CUGGCUCAUC GCUGUAGGAG 114/115/116 subunitTCCTACAGCAA UCCUACAGC AUGAGCCAG 0097 novel sequence ise2c.pk010.h3AAACAGTGCGTC ACAGUGCGUC AUAUAUUACG 117/118/119 GTAATATATTC GUAAUAUAUACGCACUGU 0098 novel sequence ise2c.pk010.h3 AAGGCACATGGT GGCACAUGGUCAGUGAAGGA 120/121/122 CCTTCACTGAT CCUUCACUG CCAUGUGCC 0099novel sequence ise2c.pk010.h3 AACACCATGACC CACCAUGACC GUACACGAGG123/124/125 CTCGTGTACAA CUCGUGUAC GUCAUGGUG 0100 Larval cuticleise2c.pk007.k24 AACGAGGCCGGA CGAGGCCGGA CUUAAGAGAU 457/458/459protein LCP-17 TCTCTTAAGCA UCUCUUAAG CCGGCCUCG 0101 Larval cuticleise2c.pk007.k24 AACTTCACACAT CUUCACACAU UGUCUAGUUA 460/461/462protein LCP-17 AACTAGACAAA AACUAGACA UGUGUGAAG 0102 Larval cuticleise2c.pk007.k24 AATGCGTGGCGA UUAGAAAUUA CUGGGCUUAU 463/464/465protein LCP-17 TTTCAAACTTA UAAGCCCAG AAUUUCUAA 0103 transcriptionalise2c.pk011.a10 AAAAAACACAGA AAAACACAGA UGAACGUGGU 126/127/128 repressorCCACGTTCACA CCACGUUCA CUGUGUUUU 0104 transcriptional ise2c.pk011.a10AATCGATGGTGG UCGAUGGUGG CGAAUAACAC 129/130/131 repressor TGTTATTCGCTUGUUAUUCG CACCAUCGA 0105 novel sequence ise2c.pk011.h12 AAAGAAAATGCTAGAAAAUGCU GUAACGCGUA 132/133/134 ACGCGTTACGA ACGCGUUAC GCAUUUUCU 0106novel sequence ise2c.pk011.h12 AACCCTTGGACA CCCUUGGACA UUCCAGUAGU135/136/137 CTACTGGAAGA CUACUGGAA GUCCAAGGG 0107 novel sequenceise2c.pk011.h12 AAGGATCCTATG GGAUCCUAUG CCUGGUACAC 138/139/140TGTACCAGGTT UGUACCAGG AUAGGAUCC 0108 translation ise2c.pk001.d22AAACTCGGCACA ACUCGGCACA AUUGUGUUGU 141/142/143 initiation  CAACACAATGGCAACACAAU GUGCCGAGU factor 5A 0109 translation ise2c.pk001.d22AATACGAAGATA UACGAAGAUA AAGGGCAGAU 144/145/146 initiation  TCTGCCCTTCCUCUGCCCUU AUCUUCGUA factor 5A 0110 translation ise2c.pk001.d22AATCAACAGCTC UCAACAGCUC UUUAUGUAAG 147/148/149 initiation  TTACATAAATGUUACAUAAA AGCUGUUGA factor 5A 0111 eukaryotic ise2c.pk001.d9AAAGAAGATCAG AGAAGAUCAG GCCAAUCUUC 150/151/152 initiation  AAGATTGGCCGAAGAUUGGC UGAUCUUCU factor eIF-4A 0112 eukaryotic ise2c.pk001.d9AAAAGCCGTCTG AAGCCGUCUG GUUGGAUAGC 153/154/155 initiation  CTATCCAACAACUAUCCAAC AGACGGCUU factor eIF-4A 0113 eukaryotic ise2c.pk001.d9AATGCTAAATGC UGCUAAAUGC GCAAGCAUGG 156/157/158 initiation  CATGCTTGCATCAUGCUUGC CAUUUAGCA factor eIF-4A 0114 Eukaryotic ise2c.pk001.i23AAGATCAGAAGA GAUCAGAAGA UCCGGCCAAU 159/160/161 initiation  TTGGCCGGAAGUUGGCCGGA CUUCUGAUC factor 4A 0115 Eukaryotic ise2c.pk001.i23AATTCTTCAGCA UUCUUCAGCA GUAUCGAUUU 162/163/164 initiation  AATCGATACCAAAUCGAUAC GCUGAAGAA factor 4A 0116 Eukaryotic ise2c.pk001.i23AAATGCTGTCAA AUGCUGUCAA UAAAUCCUCU 165/166/167 initiation  GAGGATTTAAAGAGGAUUUA UGACAGCAU factor 4A 0117 RNA ise2c.pk001.l24 AAGCTCGAGACTGCUCGAGACU UCAAGAGCAA 168/169/170 polymerase TGCTCTTGATG UGCUCUUGAGUCUCGAGC sigma subunit SigE 0118 RNA ise2c.pk001.l24 AACTGTTAGCTCCUGUUAGCUC GCAGACCUUG 171/172/173 polymerase AAGGTCTGCTA AAGGUCUGCAGCUAACAG sigma subunit SigE 0119 RNA ise2c.pk001.l24 AAGACTTTCTATGACUUUCUAU CAAAUUCUGA 174/175/176 polymerase CAGAATTTGCG CAGAAUUUGUAGAAAGUC sigma subunit SigE 0120 translation ise2c.pk005.b9AAACTTAATCAT ACUUAAUCAU UCGUCGUCCA 177/178/179 initiation  GGACGACGACAGGACGACGA UGAUUAAGU factor 2,  subunit 2 beta 0121 translationise2c.pk005.b9 AAAGAAGAAGAA AGAAGAAGAA CCCUUCUUCU 180/181/182initiation  GAAGAAGGGAG GAAGAAGGG UCUUCUUCU factor 2,  subunit 2 beta0122 translation ise2c.pk005.b9 AAGATCAAGAGA GAUCAAGAGA CCUCGACAUU183/184/185 initiation  ATGTCGAGGAT AUGUCGAGG CUCUUGAUC factor 2, subunit 2 beta 0123 putative sar1 ise2c.pk002.m10 AAAATCGTCGGTAAUCGUCGGU GUCGCUAAAA 186/187/188 protein TTTAGCGACGT UUUAGCGACCCGACGAUU 0124 putative sar1 ise2c.pk002.m10 AACTGTCAATAG CUGUCAAUAGGCAUACUGCC 189/190/191 protein GCAGTATGCGT GCAGUAUGC UAUUGACAG 0125putative sar1 ise2c.pk002.m10 AACCTGTACCAA CCUGUACCAA AGUGGUCUGU192/193/194 protein CAGACCACTGG CAGACCACU UGGUACAGG 0126 elongation ise2c.pk001.c14 AACCAAAAATGG CCAAAAAUGG UUUCCUUGCC 195/196/197factor 1-alpha GCAAGGAAAAG GCAAGGAAA CAUUUUUGG 0127 elongation ise2c.pk001.c14 AACGTGGTATCA CGUGGUAUCA UAUCGAUGGU 198/199/200factor 1-alpha CCATCGATATT CCAUCGAUA GAUACCACG 0128 elongation ise2c.pk001.c14 AACAAAATGGAC CAAAAUGGAC CUCAGUGGAG 201/202/203factor 1-alpha TCCACTGAGCC UCCACUGAG UCCAUUUUG 0129 elongationise2c.pk001.d16 AATCCGTGACTA UCCGUGACUA AUUUUUGGUU 204/205/206factor-1alpha F2  ACCAAAAATGG ACCAAAAAU AGUCACGGA 0130 elongationise2c.pk001.d16 AACATTGTCGTC CAUUGUCGUC GUGUCCAAUG 207/208/209factor-1alpha F3  ATTGGACACGT AUUGGACAC ACGACAAUG 0131 Oligosaccharylise2c.pk005.h3 AATTTGTGAGAC UUUGUGAGAC CGGCCACCAG 421/422/423transferase 48 TGGTGGCCGAA UGGUGGCCG UCUCACAAA kDa subunit 0132Oligosaccharyl ise2c.pk005.h3 AATCTGATTGTA UCUGAUUGUA GGGGGCGAAU424/425/426 transferase 48 TTCGCCCCCTC UUCGCCCCC ACAAUCAGA kDa subunit0133 Oligosaccharyl ise2c.pk005.h3 AACACTCTAGTT CACUCUAGUU AAUAGGCAGA427/428/429 transferase 48 CTGCCTATTCT CUGCCUAUU ACUAGAGUG kDa subunit0134 Myosin ise2c.pk001.d21 AACACACATCAC CACACAUCAC UCCGCCAUUG430/431/432 regulatory light AATGGCGGATA AAUGGCGGA UGAUGUGUG chain 0135Myosin ise2c.pk001.d21 AAGGATGGCATC GGAUGGCAUC CUUGCCGAUG 433/434/435regulatory light ATCGGCAAGAA AUCGGCAAG AUGCCAUCC chain 0136 Myosinise2c.pk001.d21 AAAGGCTTCATC AGGCUUCAUC CGCGGUGUCG 436/437/438regulatory light GACACCGCGAA GACACCGCG AUGAAGCCU chain 0137novel sequence ise2c.pk001.j9 AAACTCCAATTA ACUCCAAUUA AGUAGGUUAU210/211/212 TAACCTACTAG UAACCUACU AAUUGGAGU 0138 novel sequenceise2c.pk001.j9 AAGTACAAGGAT GUACAAGGAU GCCGAUCAGA 213/214/215CTGATCGGCAA CUGAUCGGC UCCUUGUAC 0139 novel sequence ise2c.pk001.j9AAGACTTTCTTC GACUUUCUUC GGGCCACAUG 216/217/218 ATGTGGCCCAT AUGUGGCCCAAGAAAGUC 0140 novel sequence ise2c.pk002.f12  AAACAAAGTATC ACAAAGUAUCGGUGUAGGCG 439/440/441 GCCTACACCGC GCCUACACC AUACUUUGU 0141novel sequence ise2c.pk002.f12  AATAGCGTCGAT UAGCGUCGAU UCGUUGAAGA442/443/444 CTTCAACGACT CUUCAACGA UCGACGCUA 0142 potassiumise2c.pk001.b14  AACTCATAGAGC CUCAUAGAGC ACACAUCAAG 219/220/221coupled amino TTGATGTGTGG UUGAUGUGU CUCUAUGAG acid transporter 0143potassium ise2c.pk001.b14 AAGATGTGGATG GAUGUGGAUG CAGUGACGUC 222/223/224coupled amino ACGTCACTGGT ACGUCACUG AUCCACAUC acid transporter  0144potassium ise2c.pk001.b14  AACCTTCCTGAT CCUUCCUGAU CAGAAGAGAA225/226/227 coupled amino TCTCTTCTGTG UCUCUUCUG UCAGGAAGGacid transporter 0145 inwardly ise2c.pk003.f2 AACAGTGCTTGT CAGUGCUUGUUCACUUAUCA 228/229/230 rectifying K+ GATAAGTGAAC GAUAAGUGA CAAGCACUGchannel protein  0146 inwardly ise2c.pk003.f2 AAGTTAATGGTG GUUAAUGGUGGAGGGCAGUC 231/232/233 rectifying K+ ACTGCCCTCGA ACUGCCCUC ACCAUUAACchannel protein  0147 inwardly ise2c.pk003.f2 AATAAAGCGATG UAAAGCGAUGCUAUGGGGUC 234/235/236 rectifying K+ ACCCCATAGGA ACCCCAUAG AUCGCUUUAchannel protein  0148 potassium ise2c.pk005.l20  AAACGGTACTGC ACGGUACUGCCUUUUUGCUG 237/238/239 coupled amino AGCAAAAAGAC AGCAAAAAG CAGUACCGUacid transporter 0149 potassium ise2c.pk005.l20  AAGCTGCATACT GCUGCAUACUGAGCCAAGAA 240/241/242 coupled amino TCTTGGCTCTC UCUUGGCUC GUAUGCAGCacid transporter 0150 potassium ise2c.pk005.l20 AAATGTTTACAG AUGUUUACAGAUCGCGUCUC 243/244/245 coupled amino AGACGCGATGA AGACGCGAU UGUAAACAUacid transporter  0151 alpha tubulin ise2c.pk001.d1 AACGTCGATCTTCGUCGAUCUU GAACUCGGUA 246/247/248 ACCGAGTTCCA ACCGAGUUC AGAUCGACG 0152tubulin alpha ise2c.pk001.k6 AATTCAAAATGC UUCAAAAUGC UGCACUCACG249/250/251 chain GTGAGTGCATC GUGAGUGCA CAUUUUGAA 0153 tubulin alphaise2c.pk001.k6 AAATCGTAGACC AUCGUAGACC CGAGGACUAG 252/253/254 chainTAGTCCTCGAC UAGUCCUCG GUCUACGAU 0154 tubulin alpha ise2c.pk001.l2AAACTCAATTCA ACUCAAUUCA CACGCAUUUU 255/256/257 chain AAATGCGTGAGAAAUGCGUG GAAUUGAGU 0155 tubulin alpha ise2c.pk001.l2 AACTTATCACTGCUUAUCACUG CUUCCUUACC 258/259/260 chain GTAAGGAAGAT GUAAGGAAG AGUGAUAAG0156 ubiquitin kinase ise2c.pk002.b4 AAGAGTTACGAA GAGUUACGAA UGGUGACGGU261/262/263 CCGTCACCATA CCGUCACCA UCGUAACUC 0157 ubiquitin kinaseise2c.pk002.b4 AAACTTAGTCCG ACUUAGUCCG UUCAUUAUCC 264/265/266GATAATGAACC GAUAAUGAA GGACUAAGU 0158 ubiquitin kinase ise2c.pk002.b4AAGGCGATGTAC GGCGAUGUAC CAGGUUCUCG 267/268/269 GAGAACCTGTT GAGAACCUGUACAUCGCC 0159 nuclear ise2c.pk001.j16  AACGACAAGATG CGACAAGAUGCUCCUUCAGC 270/271/272 ribonucleoprotein CTGAAGGAGAC CUGAAGGAG AUCUUGUCG200 kDa helicase 0160 Sqd protein ise2c.pk006.h23  AAGATAAAGGTCGAUAAAGGUC UCCACACGCG 273/274/275 homologue GCGTGTGGACC GCGUGUGGAACCUUUAUC (RNA binding) 0161 Sqd protein ise2c.pk006.h23  AATGTCAAGACTUGUCAAGACU GUUUGGAUCA 276/277/278 homologue GATCCAAACAC GAUCCAAACGUCUUGACA (RNA binding) 0162 Sqd protein ise2c.pk006.h23  AACATTCGAGTCCAUUCGAGUC ACCUGUUCAG 279/280/281 homologue TGAACAGGTGG UGAACAGGUACUCGAAUG (RNA binding) 0163 pre-mRNA- ise2c.pk006.m8 AACATAAGTCATCAUAAGUCAU UUAUUGGCCA 282/283/284 binding protein  GGCCAATAACG GGCCAAUAAUGACUUAUG 0164 pre-mRNA- ise2c.pk006.m8 AAGAACATAATA GAACAUAAUACUUCGGCACU 285/286/287 binding protein  GTGCCGAAGCC GUGCCGAAG AUUAUGUUC0165 pre-mRNA- ise2c.pk006.m8 AAACACTGGCAG ACACUGGCAG CCUCUUGAUC288/289/290 binding protein  ATCAAGAGGAT AUCAAGAGG UGCCAGUGU 0166pre-mRNA- ise2c.pk006.m8 AAGATCTTTGTT GAUCUUUGUU AAGACCACCA 291/292/293binding protein  GGTGGTCTTAG GGUGGUCUU ACAAAGAUC 0167 pre-mRNA-ise2c.pk006.m8 AACAGGTGGTCA CAGGUGGUCA GCAGCUCAUU 294/295/296binding protein  ATGAGCTGCTG AUGAGCUGC GACCACCUG 0168 chymotrypsin-ise2c.pk001.a23  AAGTTGGCTCTG GUUGGCUCUG CAAGAGUGUC 297/298/299like; protease ACACTCTTGGC ACACUCUUG AGAGCCAAC 0169 chymotrypsin-ise2c.pk001.a23  AAATCCGCAAAG AUCCGCAAAG CCUCCUCAGC 300/301/302like; protease CTGAGGAGGCA CUGAGGAGG UUUGCGGAU 0170 chymotrypsin-ise2c.pk001.a23 AACCGTGTTCTT CCGUGUUCUU AGCAGCAGUA 303/304/305like; protease ACTGCTGCTCA ACUGCUGCU AGAACACGG 0171 chymotrypsin-ise2c.pk001.a23 AACGTTGTTATG CGUUGUUAUG GCUUCCAUGC 306/307/308like; protease CATGGAAGCTG CAUGGAAGC AUAACAACG 0172 chymotrypsin-ise2c.pk001.a23 AAAACTTCGCCG AACUUCGCCG CGUUUUCACC 309/310/311like; protease GTGAAAACGCC GUGAAAACG GGCGAAGUU 0173 chymotrypsinogen;ise2c.pk001.a7  AAATGAAACTGT AUGAAACUGU CUGCGAGGAA 312/313/314 proteaseTCCTCGCAGTC UCCUCGCAG CAGUUUCAU 0174 chymotrypsinogen; ise2c.pk001.a7 AAGAAGGCTGAG GAAGGCUGAG GGUUUCUUCC 315/316/317 protease GAAGAAACCAGGAAGAAACC UCAGCCUUC 0175 chymotrypsinogen; ise2c.pk001.a7  AACTCTTTCACCCUCUUUCACC AAGUACGACG 318/319/320 protease GTCGTACTTGG GUCGUACUUGUGAAAGAG 0176 chymotrypsinogen; ise2c.pk001.a7  AACGACATTGCT CGACAUUGCUGCGGAGGACA 321/322/323 protease GTCCTCCGCAT GUCCUCCGC GCAAUGUCG 0177chymotrypsinogen; ise2c.pk001.a7  AACATCGGTACT CAUCGGUACU ACGUUGGGUA324/325/326 protease ACCCAACGTGT ACCCAACGU GUACCGAUG 0178 actin-ise2c.pk004.c4 AAGACTCAGTAC GACUCAGUAC CGAUAAUACG 327/328/329depolymerizing  GTATTATCGCG GUAUUAUCG UACUGAGUC 0179 actin-ise2c.pk004.c4  AAGTTGCCATCT GUUGCCAUCU GCAAAAGCAA 330/331/332depolymerizing TGCTTTTGCAA UGCUUUUGC GAUGGCAAC 0180 actin-ise2c.pk004.c4  AATCAGTTTGAC UCAGUUUGAC AGCGCAUGCG 333/334/335depolymerizing GCATGCGCTCA GCAUGCGCU UCAAACUGA 0181 actin-ise2c.pk004.c4  AATGGCGTCTGG UGGCGUCUGG ACUGUCACAC 336/337/338depolymerizing TGTGACAGTTT UGUGACAGU CAGACGCCA 0182 actin-ise2c.pk004.c4  AACGCGGAATAC CGCGGAAUAC GAACUGAUCG 339/340/341depolymerizing GATCAGTTCCT GAUCAGUUC UAUUCCGCG 0183 actinise2c.pk004.l4  AAGACTCAGTAC GACUCAGUAC CGAUAAUACG 342/343/344depolymerizing GTATTATCGCG GUAUUAUCG UACUGAGUC factor 0184 actinise2c.pk004.l4  AAGTTGCCATCT GUUGCCAUCU GCAAAAGCAA 345/346/347depolymerizing TGCTTTTGCAA UGCUUUUGC GAUGGCAAC factor 0185 actinise2c.pk004.l4  AATCAGTTTGAC UCAGUUUGAC AGCGCAUGCG 348/349/350depolymerizing GCATGCGCTCA GCAUGCGCU UCAAACUGA factor 0186 actinise2c.pk004.l4  AAAAATGGCGTC AAAUGGCGUC GUCACACCAG 351/352/353depolymerizing TGGTGTGACAG UGGUGUGAC ACGCCAUUU factor 0187 actinise2c.pk004.l4  AATACGATCAGT UACGAUCAGU CCUCAAGGAA 354/355/356depolymerizing TCCTTGAGGAT UCCUUGAGG CUGAUCGUA factor 0188 dismutase;ise2c.pk004.n19 AATAATTTGTGT UAAUUUGUGU UAGGGUAUGA 357/358/359superoxide CATACCCTACG CAUACCCUA CACAAAUUA 0189 dismutase;ise2c.pk004.n19 AAGTCAGTGACT GUCAGUGACU AAUUUGACCA 360/361/362superoxide GGTCAAATTCA GGUCAAAUU GUCACUGAC 0190 dismutase;ise2c.pk004.n19 AATTAGGAGATG UUAGGAGAUG CAGGUGCAAC 363/364/365superoxide TTGCACCTGGT UUGCACCUG AUCUCCUAA 0191 dismutase;ise2c.pk004.n19 AACAAACGGATG CAAACGGAUG AACCUAGGUC 366/367/368superoxide ACCTAGGTTTG ACCUAGGUU AUCCGUUUG 0192 dismutase;ise2c.pk004.n19 AATGCTGCTAGC UGCUGCUAGC AGACAUAGAG 369/370/371superoxide TCTATGTCTCC UCUAUGUCU CUAGCAGCA 0193 superoxideise2c.pk005.f21 AATTTGTGTCAT UUUGUGUCAU GCGUAGGGUA 372/373/374 dismutaseACCCTACGCAA ACCCUACGC UGACACAAA 0194 superoxide ise2c.pk005.f21AAAAGGGCTTCA AAGGGCUUCA CCUUCGACAU 375/376/377 dismutase TGTCGAAGGAGUGUCGAAGG GAAGCCCUU 0195 superoxide ise2c.pk005.f21 AATTAGGAGATGUUAGGAGAUG CAGGUGCAAC 378/379/380 dismutase TTGCACCTGGT UUGCACCUGAUCUCCUAA 0196 adenylate kinase ise2c.pk010.h5  AAAGGTCAGTAA AGGUCAGUAACACUCAUAGU 381/382/383 isozyme 3 CTATGAGTGGT CUAUGAGUG UACUGACCU 0197adenylate kinase ise2c.pk010.h5  AAGAAATATGCT GAAAUAUGCU GUGUGCCACA384/385/386 isozyme 3 GTGGCACACGT GUGGCACAC GCAUAUUUC 0198adenylate kinase ise2c.pk010.h5  AACTTGTACCTG CUUGUACCUG UGACAUCAUC387/388/389 isozyme 3 ATGATGTCATG AUGAUGUCA AGGUACAAG 0199adenylate kinase ise2c.pk010.h5  AACATTGGCTTC CAUUGGCUUC AGGAGUGUUG390/391/392 isozyme 3 AACACTCCTAA AACACUCCU AAGCCAAUG 0200adenylate kinase ise2c.pk010.h5  AAGCGGCTGGAG GCGGCUGGAG CUCAUAGAUC393/394/395 isozyme 3 ATCTATGAGAG AUCUAUGAG UCCAGCCGC 0201 ecdysoneise2c.pk001.c18 AACCCTCCCGAG CCCUCCCGAG AAUGCUCUCC 396/397/398 oxidaseGAGAGCATTAT GAGAGCAUU UCGGGAGGG 0202 ecdysone ise2c.pk001.c18AACGCCCTACGA CGCCCUACGA AAGUUCCAGU 399/400/401 oxidase CTGGAACTTCACUGGAACUU CGUAGGGCG 0203 ecdysone ise2c.pk001.c18 AACTGGACCAACCUGGACCAAC GUCCAGCACG 402/403/404 oxidase GTGCTGGACTA GUGCUGGACUUGGUCCAG 0204 innexin-2 ise2c.pk004.p1  AAAACATGGGAA AACAUGGGAAGCGACCUCCU 405/406/407 GGAGGTCGCAT GGAGGUCGC UCCCAUGUU 0205 innexin-2ise2c.pk004.p1  AAGTTGCTTGTG GUUGCUUGUG GAAGUAGUCC 408/409/410GACTACTTCCA GACUACUUC ACAAGCAAC 0206 innexin-2 ise2c.pk004.p1 AACGTCGTGGGC CGUCGUGGGC GAAGAUCUGG 411/412/413 CAGATCTTCTT CAGAUCUUCCCCACGACG 0207 innexin-2 ise2c.pk004.p1  AATACGGTCCTT UACGGUCCUUCGGUUCCUGA 415/416/417 CAGGAACCGTG CAGGAACCG AGGACCGUA 0208 innexin-2ise2c.pk004.p1 AATTTCGCTGAT UUUCGCUGAU AUGCGGUAAA 418/419/420TTACCGCATGG UUACCGCAU UCAGCGAAA (Note: the sense RNA primer sequence andthe antisense RNA primer sequences shown in table 2 were generated with2 thymine residues at the 3′ end.)Droplet Feeding Assay for Evaluation of 21mer dsRNA InsecticidalProperties Against the Fall Armyworm Spodoptera frugiperda

10 nanoMole quantities of 21mer desalted primers were purchased fromProligo (Sigma Aldrich, St. Louis, Miss.). The lyophilized sample issolubilized in nuclease free water at a 100 uMolar concentration. Thestock solution was then diluted in 20% sucrose containing blue McCormickfood coloring. O.5 ul droplets of this solution were dispensed in acircle in a parafilm-lined 65 mm petridish. Sucrose blanks were used ascontrols. Between 20 and 30 neonate fall armyworms were then added tothe middle of the droplet circle and the petri dish sealed withparafilm. After two hours, the neonates with blue digestive tracts wereremoved and placed on standard multispecies lepidopteran insect diet.Insects were evaluated at 48, 72, and 96 hours post challenge formortality and growth inhibition.

Serial dilution assays starting with a high dose of 20 uM and including10, 5, 2.5, 1.25, 0.6, and 0 uMolar concentrations were also performedin this manner.

TABLE 3 Rep 1 Rep 2 72 H 72 H Primer Insects ave. Insects ave. Combined# Target gene treated weight treated weight ave. 75 juvenile hormonediol kinase 9 10 11 11 11 83 juvenile hormone diol kinase 15 14 14 12 1391 V-ATPase A subunit 14 14 11 15 14 99 conserved hypothetical 16 15 1916 16 protein 107 novel sequence (cuticular 16 14 13 15 14 protein?) 115Eukaryotic initiation factor 4A 16 9 18 14 12 123 putative sar1 protein16 15 16 17 16 131 Oligosaccharyl transferase 17 13 14 15 14 48 kDasubunit 139 myosin protein 13 15 16 18 17 147 inwardly rectifying K+ 1511 15 16 14 channel protein 155 alpha tubulin chain 15 10 16 19 15sucrose control 14 17 15 18 18Sucrose Droplet Feeding Assay.

Neonate larvae were fed 25 uMolar dsRNAs. Treated insects were weigheden masse at 72 hours and compared to sucrose controls. 2 replicates ofthe experiment were averaged.

Injection Feeding Assay for Evaluation of 21mer dsRNA InsecticidalProperties Against the Fall Armyworm Spodoptera frugiperda

Second instar fall armyworm were injected using a micromanipulator andmicroinjection needles pulled on a Sutter Instrument (Novato, Calif.)P-2000 horizontal needle puller. The needle was back loaded with dsRNAsolution. Initial injection experiments employed a concentration of 2ug/ul (see Table X). This rate produced high mortality across allprimers tested. Subsequent assays were performed with lowerconcentrations. Blue McCormick food coloring was included in the dsRNAsolution to better visualize the injection process. Prior to injection,the insects were affixed to a microscope slide using a glue stick(Office Depot, Delray Beach, Fla.). The injection needle was connectedto a 20 ml hypodermic syringe via Teflon tubing. The injection needlewas then mounted on a Leitz micromanipulator. The dsRNA solution wasdispensed from the microinjection needle by pressing on the plunger ofthe 20 ml syringe. Injection volumes were variable but averagedapproximately 250 nL (based on injection of approximately 20 insectsinjected from a 5 ul volume loaded into the needle). Followinginjection, insects were removed from the microscope slide with the aidof a moistened fine camelhair brush. The insects were then placed onmultispecies diet and were evaluated for mortality at 24 and 48 hours.Water injections were used as controls. Silencer® Negative Control #1,2, and 3 siRNA control primers from Ambion (Austin, Tex.) were alsoincluded as negative controls.

TABLE 4 Primer No. # Target gene injected Alive Dead 75 juvenile hormonediol kinase 6 0 6 83 juvenile hormone diol kinase 6 0 6 91 V-ATPase Asubunit 8 3 5 99 conserved hypothetical sequence 8 0 8 107 novelsequence (cuticular protein 8 0 8 115 Eukaryotic initiation factor 4A 80 8 123 putative sar1 protein 8 1 8 131 Oligosaccharyl transferase 8 2 848 kDa subunit 147 inwardly rectifying K+ 8 1 7 channel protein Water 87 1Microinjection of dsRNAs [2 ug/ul].

TABLE 5 Microinjection of dsRNAs [0.7 ug/ul] into FAW neonate larvae Rep1 Rep 2 Primer # Target gene # Injected 24 H dead 48 H dead # Injected24 H dead 48 H dead 75 juvenile hormone diol kinase 1 7 2 3 11 7 7 83juvenile hormone diol kinase 2 11 10 10 12 6 5 91 V-ATPase A subunit 139 7 9 9 9 99 conserved hypothetical protein 1 9 2 2 107 novel sequence(cuticular protein?) 11 5 6 115 Eukaryotic initiation factor 4A 8 6 6123 putative sar1 protein 13 9 9 131 Oligosaccharyl transferase 48 kDasubunit 8 6 4 139 myosin protein 10 4 4 147 inwardly rectifying K+channel protein 14 8 9 155 alpha tubulin chain 11 9 9 Ambion controlprimer 1 6 0 0 11 0 0 Ambion control primer 2 11 0 0 8 0 0 Ambioncontrol primer 3 12 1 1 9 1 1Microinjection Assay using 0.7 ug/ul dose of dsRNA 21mers. Note; On someoccasions, the mortality was lower at 48 hours than at 24 hours. This isdue to moribund insects recovering at the later time point.Topical Diet Assay for Evaluation of 21Mer dsRNA Insecticidal PropertiesAgainst the Fall Armyworm Spodoptera frugiperda

The term “topical diet assay” refers to assays where artificial dietsare pipetted into microtiter plates and the dsRNA solution is dispensedon the surface of the diet. In the dsRNA experiments, 100 ul of diet wasdispensed per well. The surface of the well was then treated with 10 ulof a dsRNA solution of varying concentrations. The plates were theninfested with 1 neonate fall armyworm per well and sealed with mylar.The mylar seal was punctured with a small insect pin to allow for airexchange. Plates were then stored in a growth chamber at 28 C and theassay was scored for stunting or mortality at 4 days. Table 6-12represents several experiments using this method. Table 13 provides asummary of the data.

In topical assay #1, the primers that previously showed activity ininjection assays were tested in a FAW topical diet assay. These resultsare shown in Table 6. A 50 uMolar solution (0.66 ug/ul) was used as thetest concentration. 5 ul of this sample was loaded onto the top of 100ul of diet producing a final concentration of 2.5 uMolar or 30 ppm. Inaddition to A1-A11 (A12 is a negative control), the other samples arethose with no known human orthologs. The plate was infested with aprox.5 neonates/well. The scoring period was 72 hours.

In topical assay #2, primers were tested in a FAW topical diet assay,and the results are shown in table 7. In this experiment, the 2.7 ug/ulstock was diluted to a starting concentration of 0.67 ug/ul. 2 foldserial dilution was carried out to produce stocks of 0.32 ug/ul and 0.16ug/ul. 5 ul of these stocks were added to the 100 ul of diet producingfinal concentrations of 30, 15, and 8 ppm in diet. The scoring periodwas 72 hours.

In topical assay #3, primers were tested in a FAW topical diet assay,and the results are shown in table 8. In this experiment, the 2.7 ug/ulstock was diluted to a starting concentration of 0.67 ug/ul. 2 foldserial dilution was carried out to produce stocks of 0.32 ug/ul and 0.16ug/ul. 5 ul of these stocks were added to the 100 ul of diet producingfinal concentrations of 30, 15, and 8 ppm in diet. This is a replicateof the previous experiment. The scoring period was 72 hours.

In topical assay #4, primers were tested in a FAW topical diet assay,and the results are shown in table 9. In this experiment, the 2.7 ug/ulstock was diluted to a starting concentration of 0.67 ug/ul. 2 foldserial dilution was carried out to produce stocks of 0.32 ug/ul and 0.16ug/ul. 5 ul of these stocks were added to the 100 ul of diet producingfinal concentrations of 30, 15, and 8 ppm in diet. The scoring periodwas 72 hours.

A summary of the topical assay data shown in tables 6-9 appears in Table10.

In topical assay #5, primers were tested in a FAW topical diet assay andthe results are shown in Table 11. 50 ul of 0.16 ug/ul primers weremixed with 50 ul water and then serially diluted. 10 ul of the samplethen added to the wells. Therefore the first concentration was 10ul×0.08 ug/ul=0.8 ug total dsRNA/100 ul diet=8 ppm. This was ½ the rateof previous experiments (assays #1-4) where 5 ul of 0.32 showedactivity. The scoring period was 72 hours. A score of “S” indicatesclear stunting compared to untreated controls. A score of “ss” indicateslive insects but exhibiting severe stunting defined as little or nogrowth beyond the neonate body size.

In topical assay #6, primers were tested in a FAW topical diet assay andthe results are shown in Table 12. The first rate is 10 ul of the 0.16ug/ul primer stock. From there, 50 ul of 0.16 ug/ul primer mixed with 50ul water and then serially diluted. 10 ul of the sample was then addedto the wells. Therefore first concentration was 10 ul×0.16 ug/ul=1.6 ugtotal dsRNA/100 ul diet=16 ppm. The scoring period was 72 hours.

TABLE 6 SEQ ID NO Target 30 Target region/ Sample seq id gene idsequence forward reverse ppm sense/antisense 0075 ise1c.pk002.m13 Juvenile AACATGGTATCC CAUGGUAUCC CCUGAAGUCG 51/52/53 hormone GACTTCAGGAAGACUUCAGG GAUACCAUG query 0083 ise1c.pk005.a15 Juvenile AAGTTCGCGTTCGUUCGCGUUC UUCAAGAGUG + 75/76/77 hormone ACTCTTGAAGA ACUCUUGAA AACGCGAACquery 0085 ise1c.pk006.d24  Juvenile AATCACGCTGAA UCACGCUGAAUACAGUGGUU + 81/82/83 hormone ACCACTGTATA ACCACUGUA UCAGCGUGA query 0086ise2c.pk009.i4 Juvenile AAAATATGGCGC AAUAUGGCGC ACAAUAGGCG 84/85/86hormone GCCTATTGTTT GCCUAUUGU CGCCAUAUU query 0088 ise2c.pk009.i5Juvenile AAGTCATCGTTC GUCAUCGUUC GUAGACUUGG + 90/91/92 hormoneCAAGTCTACCT CAAGUCUAC AACGAUGAC query 0089 ise2c.pk001.d19 vacuolarAACCCCTTGAAT CCCCUUGAAU GACCUUAACA + 93/94/95 query GTTAAGGTCGGGUUAAGGUC UUCAAGGGG 0091 ise2c.pk001.d20 vacuolar AACGTGTCCATGCGUGUCCAUG GUCAGCCAUC +  99/100/101 query ATGGCTGACTC AUGGCUGACAUGGACACG 0094 ise2c.pk001.f20 vacuolar AACTCTGACGTC CUCUGACGUCGUAGAUGAUG + 108/109/110 query ATCATCTACGT AUCAUCUAC ACGUCAGAG 0095ise2c.pk001.f21 vacuolar AAGTGCTTGGGT GUGCUUGGGU GUCGGGGUUA +111/112/113 query AACCCCGACAG AACCCCGAC CCCAAGCAC 0099 ise2c.pk010.h3cadherin AACACCATGACC CACCAUGACC GUACACGAGG 123/124/125 queryCTCGTGTACAA CUCGUGUAC GUCAUGGUG 0107 ise2c.pkOl1.h12 cuticleAAGGATCCTATG GGAUCCUAUG CCUGGUACAC + 138/139/140 protein TGTACCAGGTTUGUACCAGG AUAGGAUCC 0115 ise2c.pk001.i23 Translation AATTCTTCAGCAUUCUUCAGCA GUAUCGAUUU 162/163/164 initiation AATCGATACCA AAUCGAUACGCUGAAGAA factor 0123 ise2c.pk002.m10 SAR1 AAAATCGTCGGT AAUCGUCGGUGUCGCUAAAA 186/187/188 TTTAGCGACGT UUUAGCGAC CCGACGAUU 0131ise2c.pk005.h3  phosphooligo- AATTTGTGAGAC UUUGUGAGAC CGGCCACCAG421/422/423 saccharide TGGTGGCCGAA UGGUGGCCG UCUCACAAA . . . 0139ise2c.pk001.j9  Myosin AAGACTTTCTTC GACUUUCUUC GGGCCACAUG 216/217/218ATGTGGCCCAT AUGUGGCCC AAGAAAGUC 0147 ise2c.pk003.f2  PotassiumAATAAAGCGATG UAAAGCGAUG CUAUGGGGUC + 234/235/236 inwardly ACCCCATAGGAACCCCAUAG AUCGCUUUA rectifying protein 0155 ise2c.pk001.l2  TubulinAACTTATCACTG CUUAUCACUG CUUCCUUACC 258/259/260 GTAAGGAAGAT GUAAGGAAGAGUGAUAAG (Note: the sense RNA primer sequence and the antisen RNAprimer sequences shown in table 6 were generated with 2 thymine residuesat the 3' end.)

TABLE 7  SEQ ID NO Target 30 Target region/ Sample seq id gene idsequence forward reverse ppm sense/antisense 0075 ise1c.pk002.m13Juvenile AACATGGTATCC CAUGGUAUCC CCUGAAGUCG 51/52/53 hormone GACTTCAGGAAGACUUCAGG GAUACCAUG query 0076 AAGGTCGCTGAC GGUCGCUGAC CUUGUUCUCG54/55/56 GAGAACAAGGA GAGAACAAG UCAGCGACC 0077 AAGTGTCCTGGG GUGUCCUGGGGAACUCAAGC 57/58/59 CTTGAGTTCCA CUUGAGUUC CCAGGACAC 0078 ise1c.pk003.f7Juvenile AAGAAGAAGCTC GAAGAAGCUC CACGUGGAGG 60/61/62 hormone CTCCACGTGTTCUCCACGUG AGCUUCUUC query 0079 AAGGTCGCTGAC GGUCGCUGAC CUUGUUCUCG63/64/65 GAGAACAAGGA GAGAACAAG UCAGCGACC 0080 AATGTCCTGGGG UGUCCUGGGGGAAACUCAGC 66/67/68 CTGAGTTTCAA CUGAGUUUC CCCAGGACA 0081 ise1c.pk005.a15Juvenile AAGAATAAGCTC GAAUAAGCUC CACGUGGAGG 69/70/71 hormone CTCCACGTGTTCUCCACGUG AGCUUAUUC query 0082 AATTTGTCGAGG UUUGUCGAGG AUAGGGUCUC72/73/74 AGACCCTATTG AGACCCUAU CUCGACAAA 0083 AAGTTCGCGTTC GUUCGCGUUCUUCAAGAGUG + 75/76/77 ACTCTTGAAGA ACUCUUGAA AACGCGAAC 0084ise1c.pk006.d24  Juvenile AACTGCCCCTTA CUGCCCCUUA AGAUGAGGUU 78/79/80hormone ACCTCATCTAT ACCUCAUCU AAGGGGCAG query 0085 AATCACGCTGAAUCACGCUGAA UACAGUGGUU 81/82/83 ACCACTGTATA ACCACUGUA UCAGCGUGA 0086ise2c.pk009.i4  Juvenile AAAATATGGCGC AAUAUGGCGC ACAAUAGGCG 84/85/86hormone GCCTATTGTTT GCCUAUUGU CGCCAUAUU query 0087 AACGTTCTCGGTCGUUCUCGGU CAGUGAAAGA 87/88/89 CTTTCACTGCT CUUUCACUG CCGAGAACG 0088AAGTCATCGTTC GUCAUCGUUC GUAGACUUGG 90/91/92 CAAGTCTACCT CAAGUCUACAACGAUGAC 0089 ise2c.pk001.d19  vacuolar AACCCCTTGAAT CCCCUUGAAUGACCUUAACA 93/94/95 query GTTAAGGTCGG GUUAAGGUC UUCAAGGGG 0090AAGTACACCATG GUACACCAUG UACUUGCAAC 96/97/98 TTGCAAGTATG UUGCAAGUAAUGGUGUAC 0091 AACGTGTCCATG CGUGUCCAUG GUCAGCCAUC  99/100/101ATGGCTGACTC AUGGCUGAC AUGGACACG 0092 ise2c.pk001.e14  vacuolarAAACCTACAAAA ACCUACAAAA UUUCGGCCAU 102/103/104 query TGGCCGAAAACUGGCCGAAA UUUGUAGGU 0093 AATCTACGGACC UCUACGGACC CCAAAGAAGG 105/106/107CTTCTTTGGAG CUUCUUUGG GUCCGUAGA 0094 ise2c.pk001.f20  vacuolarAACTCTGACGTC CUCUGACGUC GUAGAUGAUG 108/109/110 query ATCATCTACGTAUCAUCUAC ACGUCAGAG 0095 AAGTGCTTGGGT GUGCUUGGGU GUCGGGGUUA 111/112/113AACCCCGACAG AACCCCGAC CCCAAGCAC 0096 AACTGGCTCATC CUGGCUCAUC GCUGUAGGAG114/115/116 TCCTACAGCAA UCCUACAGC AUGAGCCAG 0097 ise2c.pk010.h3 cadherin AAACAGTGCGTC ACAGUGCGUC AUAUAUUACG 117/118/119 queryGTAATATATTC GUAAUAUAU ACGCACUGU 0098 AAGGCACATGGT GGCACAUGGUCAGUGAAGGA + 120/121/122 CCTTCACTGAT CCUUCACUG CCAUGUGCC 0099AACACCATGACC CACCAUGACC GUACACGAGG 123/124/125 CTCGTGTACAA CUCGUGUACGUCAUGGUG 0100 ise2c.pk007.k24  cuticle AACGAGGCCGGA CGAGGCCGGACUUAAGAGAU 457/458/459 protein TCTCTTAAGCA UCUCUUAAG CCGGCCUCG 0101AACTTCACACAT CUUCACACAU UGUCUAGUUA 460/461/462 AACTAGACAAA AACUAGACAUGUGUGAAG 0102 AATGCGTGGCGA UUAGAAAUUA CUGGGCUUAU 463/464/465TTTCAAACTTA UAAGCCCAG AAUUUCUAA 0103 ise2c.pk011.a10  cuticleAAAAAACACAGA AAAACACAGA UGAACGUGGU + 126/127/128 protein CCACGTTCACACCACGUUCA CUGUGUUUU 0104 AATCGATGGTGG UCGAUGGUGG CGAAUAACAC +129/130/131 TGTTATTCGCT UGUUAUUCG CACCAUCGA 0105 ise2c.pk011.h12 cuticle AAAGAAAATGCT AGAAAAUGCU GUAACGCGUA 132/133/134 proteinACGCGTTACGA ACGCGUUAC GCAUUUUCU 0106 AACCCTTGGACA CCCUUGGACAUUCCAGUAGU + 135/136/137 CTACTGGAAGA CUACUGGAA GUCCAAGGG 0107AAGGATCCTATG GGAUCCUAUG CCUGGUACAC 138/139/140 TGTACCAGGTT UGUACCAGGAUAGGAUCC 0108 ise2c.pk001.d22  translation  AAACTCGGCACA ACUCGGCACAAUUGUGUUGU 141/142/143 initiation CAACACAATGG CAACACAAU GUGCCGAGU factor0109 AATACGAAGATA UACGAAGAUA AAGGGCAGAU + 144/145/146 TCTGCCCTTCCUCUGCCCUU AUCUUCGUA 0110 AATCAACAGCTC UCAACAGCUC UUUAUGUAAG 147/148/149TTACATAAATG UUACAUAAA AGCUGUUGA 0111 ise2c.pk001.d9  translation AAAGAAGATCAG AGAAGAUCAG GCCAAUCUUC 150/151/152 initiation AAGATTGGCCGAAGAUUGGC UGAUCUUCU factor 0112 AAAAGCCGTCTG AAGCCGUCUG GUUGGAUAGC +153/154/155 CTATCCAACAA CUAUCCAAC AGACGGCUU 0113 AATGCTAAATGC UGCUAAAUGCGCAAGCAUGG 156/157/158 CATGCTTGCAT CAUGCUUGC CAUUUAGCA 0114ise2c.pk001.i23  translation  AAGATCAGAAGA GAUCAGAAGA UCCGGCCAAU +159/160/161 initiation TTGGCCGGAAG UUGGCCGGA CUUCUGAUC factor 0115AATTCTTCAGCA UUCUUCAGCA GUAUCGAUUU 162/163/164 AATCGATACCA AAUCGAUACGCUGAAGAA 0116 AAATGCTGTCAA AUGCUGUCAA UAAAUCCUCU 165/166/167GAGGATTTAAA GAGGAUUUA UGACAGCAU 0117 ise2c.pk001.l24  translation AAGCTCGAGACT GCUCGAGACU UCAAGAGCAA 168/169/170 initiation TGCTCTTGATGUGCUCUUGA GUCUCGAGC factor 0118 AACTGTTAGCTC CUGUUAGCUC GCAGACCUUG171/172/173 AAGGTCTGCTA AAGGUCUGC AGCUAACAG 0119 AAGACTTTCTAT GACUUUCUAUCAAAUUCUGA + 174/175/176 CAGAATTTGCG CAGAAUUUG UAGAAAGUC 0120ise2c.pk005.b9  translation  AAACTTAATCAT ACUUAAUCAU UCGUCGUCCA177/178/179 initiation GGACGACGACA GGACGACGA UGAUUAAGU factor 0121AAAGAAGAAGAA AGAAGAAGAA CCCUUCUUCU + 180/181/182 GAAGAAGGGAG GAAGAAGGGUCUUCUUCU 0122 AAGATCAAGAGA GAUCAAGAGA CCUCGACAUU + 183/184/185ATGTCGAGGAT AUGUCGAGG CUCUUGAUC 0123 ise2c.pk002.m10 SARI AAAATCGTCGGTAAUCGUCGGU GUCGCUAAAA 186/187/188 TTTAGCGACGT UUUAGCGAC CCGACGAUU 0124AACTGTCAATAG CUGUCAAUAG GCAUACUGCC 189/190/191 GCAGTATGCGT GCAGUAUGCUAUUGACAG 0125 AACCTGTACCAA CCUGUACCAA AGUGGUCUGU + 192/193/194CAGACCACTGG CAGACCACU UGGUACAGG 0126 ise2c.pk001.c14  ElongationAACCAAAAATGG CCAAAAAUGG UUUCCUUGCC + 195/196/197 factor GCAAGGAAAAGGCAAGGAAA CAUUUUUGG 0127 AACGTGGTATCA CGUGGUAUCA UAUCGAUGGU +198/199/200 CCATCGATATT CCAUCGAUA GAUACCACG 0128 AACAAAATGGAC CAAAAUGGACCUCAGUGGAG 201/202/203 TCCACTGAGCC UCCACUGAG UCCAUUUUG 0129ise2c.pk001.d16 Elongation AATCCGTGACTA UCCGUGACUA AUUUUUGGUU +204/205/206 factor ACCAAAAATGG ACCAAAAAU AGUCACGGA 0130 AACATTGTCGTCCAUUGUCGUC GUGUCCAAUG 207/208/209 ATTGGACACGT AUUGGACAC ACGACAAUG 0131ise2c.pk005.h3  phosphooligo- AATTTGTGAGAC UUUGUGAGAC CGGCCACCAG421/422/423 saccharide  TGGTGGCCGAA UGGUGGCCG UCUCACAAA . . . 0132AATCTGATTGTA UCUGAUUGUA GGGGGCGAAU 424/245/426 TTCGCCCCCTC UUCGCCCCCACAAUCAGA 0133 AACACTCTAGTT CACUCUAGUU AAUAGGCAGA 427/228/429CTGCCTATTCT CUGCCUAUU ACUAGAGUG 0134 ise2c.pk001.d21 myosin AACACACATCACCACACAUCAC UCCGCCAUUG 430/431/432 AATGGCGGATA AAUGGCGGA UGAUGUGUG 0135AAGGATGGCATC GGAUGGCAUC CUUGCCGAUG 433/434/435 ATCGGCAAGAA AUCGGCAAGAUGCCAUCC 0136 AAAGGCTTCATC AGGCUUCAUC CGCGGUGUCG 436/437/438GACACCGCGAA GACACCGCG AUGAAGCCU 0137 ise2c.pk001.j9 myosin AAACTCCAATTAACUCCAAUUA AGUAGGUUAU 210/211/212 TAACCTACTAG UAACCUACU AAUUGGAGU 0138AAGTACAAGGAT GUACAAGGAU GCCGAUCAGA + 213/214/215 CTGATCGGCAA CUGAUCGGCUCCUUGUAC 0139 AAGACTTTCTTC GACUUUCUUC GGGCCACAUG 216/217/218ATGTGGCCCAT AUGUGGCCC AAGAAAGUC 0140 ise2c.pk002.f12 myosin AAACAAAGTATCACAAAGUAUC GGUGUAGGCG 439/440/441 GCCTACACCGC GCCUACACC AUACUUUGU 0141AATAGCGTCGAT UAGCGUCGAU UCGUUGAAGA 442/443/444 CTTCAACGACT CUUCAACGAUCGACGCUA 0142 ise2c.pk001.b14   potassium AACTCATAGAGC CUCAUAGAGCACACAUCAAG 219/220/221 channel TTGATGTGTGG UUGAUGUGU CUCUAUGAGamino acid transporter 0143 AAGATGTGGATG GAUGUGGAUG CAGUGACGUC221/223/224 ACGTCACTGGT ACGUCACUG AUCCACAUC 0144 AACCTTCCTGAT CCUUCCUGAUCAGAAGAGAA 225/226/227 TCTCTTCTGTG UCUCUUCUG UCAGGAAGG 0145ise2c.pk003.f2 potassium AACAGTGCTTGT CAGUGCUUGU UCACUUAUCA +228/229/230 inwardly GATAAGTGAAC GAUAAGUGA CAAGCACUG rectifier . . .0146 AAGTTAATGGTG GUUAAUGGUG GAGGGCAGUC + 231/232/233 ACTGCCCTCGAACUGCCCUC ACCAUUAAC 0147 AATAAAGCGATG UAAAGCGAUG CUAUGGGGUC +234/235/236 ACCCCATAGGA ACCCCAUAG AUCGCUUUA 0148 ise2c.pk005.l20amino acid AAACGGTACTGC ACGGUACUGC CUUUUUGCUG + 237/238/239 transporterAGCAAAAAGAC AGCAAAAAG CAGUACCGU 0149 AAGCTGCATACT GCUGCAUACUGAGCCAAGAA + 240/241/242 TCTTGGCTCTC UCUUGGCUC GUAUGCAGC 0150AAATGTTTACAG AUGUUUACAG AUCGCGUCUC 243/244/245 AGACGCGATGA AGACGCGAUUGUAAACAU 0151 ise2c.pk001.d1 tubulin AACGTCGATCTT CGUCGAUCUUGAACUCGGUA + 246/247/248 ACCGAGTTCCA ACCGAGUUC AGAUCGACG 0152ise2c.pk001.k6 tubulin AATTCAAAATGC UUCAAAAUGC UGCACUCACG 249/250/251GTGAGTGCATC GUGAGUGCA CAUUUUGAA 0153 AAATCGTAGACC AUCGUAGACCCGAGGACUAG + 252/253/254 TAGTCCTCGAC UAGUCCUCG GUCUACGAU 0154ise2c.pk001.l2 tubulin AAACTCAATTCA ACUCAAUUCA CACGCAUUUU + 255/256/257AAATGCGTGAG AAAUGCGUG GAAUUGAGU 0155 AACTTATCACTG CUUAUCACUG CUUCCUUACC258/259/260 GTAAGGAAGAT GUAAGGAAG AGUGAUAAG 0156 ise2c.pk002.b4ubiquitin AAGAGTTACGAA GAGUUACGAA UGGUGACGGU 261/262/263 CCGTCACCATACCGUCACCA UCGUAACUC 0157 AAACTTAGTCCG ACUUAGUCCG UUCAUUAUCC +264/265/266 GATAATGAACC GAUAAUGAA GGACUAAGU 0158 AAGGCGATGTAC GGCGAUGUACCAGGUUCUCG + 267/268/269 GAGAACCTGTT GAGAACCUG UACAUCGCC 0159ise2c.pk001.j16 small AACGACAAGATG CGACAAGAUG CUCCUUCAGC + 270/271/272nuclear CTGAAGGAGAC CUGAAGGAG AUCUUGUCG ribonucleo- protein 0160ise2c.pk006.h23 small AAGATAAAGGTC GAUAAAGGUC UCCACACGCG 273/274/275nuclear GCGTGTGGACC GCGUGUGGA ACCUUUAUC ribonucleo- protein 0161AATGTCAAGACT  UGUCAAGACU GUUUGGAUCA 276/277/278 GATCCAAACAC GAUCCAAACGUCUUGACA 0162 AACATTCGAGTC CAUUCGAGUC ACCUGUUCAG + 279/280/281TGAACAGGTGG UGAACAGGU ACUCGAAUG (Note: the sense RNA primer sequence andthe antisense RNA primer sequences shown in table 7 were generated with2 thymine residues at the 3′ end.)

TABLE 8 SEQ ID NO Target 30 Target region/ Sample seq id gene idsequence forward reverse ppm sense/antisense 0075 ise1c.pk002.m13Juvenile AACATGGTATCC CAUGGUAUCC CCUGAAGUCG 51/52/53 hormone GACTTCAGGAAGACUUCAGG GAUACCAUG query 0076 AAGGTCGCTGAC GGUCGCUGAC CUUGUUCUCG54/55/56 GAGAACAAGGA GAGAACAAG UCAGCGACC 0077 AAGTGTCCTGGG GUGUCCUGGGGAACUCAAGC 57/58/59 CTTGAGTTCCA CUUGAGUUC CCAGGACAC 0078 ise1c.pk003.f7Juvenile AAGAAGAAGCTC GAAGAAGCUC CACGUGGAGG 60/61/62 hormone CTCCACGTGTTCUCCACGUG AGCUUCUUC query 0079 AAGGTCGCTGAC GGUCGCUGAC CUUGUUCUCG63/64/65 GAGAACAAGGA GAGAACAAG UCAGCGACC 0080 AATGTCCTGGGG UGUCCUGGGGGAAACUCAGC + 66/67/68 CTGAGTTTCAA CUGAGUUUC CCCAGGACA 0081ise1c.pk005.a15 Juvenile AAGAATAAGCTC GAAUAAGCUC CACGUGGAGG 69/70/71hormone CTCCACGTGTT CUCCACGUG AGCUUAUUC query 0082 AATTTGTCGAGGUUUGUCGAGG AUAGGGUCUC 72/73/74 AGACCCTATTG AGACCCUAU CUCGACAAA 0083AAGTTCGCGTTC GUUCGCGUUC UUCAAGAGUG NT 75/76/77 ACTCTTGAAGA ACUCUUGAAAACGCGAAC 0084 ise1c.pk006.d24 Juvenile AACTGCCCCTTA CUGCCCCUUAAGAUGAGGUU + 78/79/80 hormone ACCTCATCTAT ACCUCAUCU AAGGGGCAG query 0085AATCACGCTGAA UCACGCUGAA UACAGUGGUU 81/82/83 ACCACTGTATA ACCACUGUAUCAGCGUGA 0086 ise2c.pk009.i4 Juvenile AAAATATGGCGC AAUAUGGCGCACAAUAGGCG 84/85/86 hormone GCCTATTGTTT GCCUAUUGU CGCCAUAUU query 0087AACGTTCTCGGT CGUUCUCGGU CAGUGAAAGA 87/88/89 CTTTCACTGCT CUUUCACUGCCGAGAACG 0088 AAGTCATCGTTC GUCAUCGUUC GUAGACUUGG 90/91/92 CAAGTCTACCTCAAGUCUAC AACGAUGAC 0089 ise2c.pk001.d19 vacuolar AACCCCTTGAATCCCCUUGAAU GACCUUAACA + 93/94/95 query GTTAAGGTCGG GUUAAGGUC UUCAAGGGG0090 AAGTACACCATG GUACACCAUG UACUUGCAAC 96/97/98 TTGCAAGTATG UUGCAAGUAAUGGUGUAC 0091 AACGTGTCCATG CGUGUCCAUG GUCAGCCAUC +  99/100/101ATGGCTGACTC AUGGCUGAC AUGGACACG 0092 ise2c.pk001.e14 vacuolarAAACCTACAAAA ACCUACAAAA UUUCGGCCAU 102/103/104 query TGGCCGAAAACUGGCCGAAA UUUGUAGGU 0093 AATCTACGGACC UCUACGGACC CCAAAGAAGG 105/106/107CTTCTTTGGAG CUUCUUUGG GUCCGUAGA 0094 ise2c.pk001.f20 vacuolarAACTCTGACGTC CUCUGACGUC GUAGAUGAUG 108/109/110 query ATCATCTACGTAUCAUCUAC ACGUCAGAG 0095 AAGTGCTTGGGT GUGCUUGGGU GUCGGGGUUA 111/112/113AACCCCGACAG AACCCCGAC CCCAAGCAC 0096 AACTGGCTCATC CUGGCUCAUC GCUGUAGGAG114/115/116 TCCTACAGCAA UCCUACAGC AUGAGCCAG 0097 ise2c.pk010.h3 cadherinAAACAGTGCGTC ACAGUGCGUC AUAUAUUACG 117/118/119 query GTAATATATTCGUAAUAUAU ACGCACUGU 0098 AAGGCACATGGT GGCACAUGGU CAGUGAAGGA 120/121/122CCTTCACTGAT CCUUCACUG CCAUGUGCC 0099 AACACCATGACC CACCAUGACC GUACACGAGG123/124/125 CTCGTGTACAA CUCGUGUAC GUCAUGGUG 0100 ise2c.pk007.k24 cuticleAACGAGGCCGGA CGAGGCCGGA CUUAAGAGAU 457/458/459 protein TCTCTTAAGCAUCUCUUAAG CCGGCCUCG 0101 AACTTCACACAT CUUCACACAU UGUCUAGUUA 460/461/462AACTAGACAAA AACUAGACA UGUGUGAAG 0102 AATGCGTGGCGA UUAGAAAUUA CUGGGCUUAU463/464/465 TTTCAAACTTA UAAGCCCAG AAUUUCUAA 0103 ise2c.pk011.a10 cuticleAAAAAACACAGA AAAACACAGA UGAACGUGGU 126/127/128 protein CCACGTTCACACCACGUUCA CUGUGUUUU 0104 AATCGATGGTGG UCGAUGGUGG CGAAUAACAC +129/130/131 TGTTATTCGCT UGUUAUUCG CACCAUCGA 0105 ise2c.pk011.h12 cuticleAAAGAAAATGCT AGAAAAUGCU GUAACGCGUA 132/133/134 protein ACGCGTTACGAACGCGUUAC GCAUUUUCU 0106 AACCCTTGGACA CCCUUGGACA UUCCAGUAGU 135/136/137CTACTGGAAGA CUACUGGAA GUCCAAGGG 0107 AAGGATCCTATG GGAUCCUAUG CCUGGUACAC138/139/140 TGTACCAGGTT UGUACCAGG AUAGGAUCC 0108 ise2c.pk001.d22translation AAACTCGGCACA ACUCGGCACA AUUGUGUUGU + 141/142/143 initiationCAACACAATGG CAACACAAU GUGCCGAGU factor 0109 AATACGAAGATA UACGAAGAUAAAGGGCAGAU + 144/145/146 TCTGCCCTTCC UCUGCCCUU AUCUUCGUA 0110AATCAACAGCTC UCAACAGCUC UUUAUGUAAG + 147/148/149 TTACATAAATG UUACAUAAAAGCUGUUGA 0111 ise2c.pk001.d9 translation AAAGAAGATCAG AGAAGAUCAGGCCAAUCUUC 150/151/152 initiation AAGATTGGCCG AAGAUUGGC UGAUCUUCU factor0112 AAAAGCCGTCTG AAGCCGUCUG GUUGGAUAGC 153/154/155 CTATCCAACAACUAUCCAAC AGACGGCUU 0113 AATGCTAAATGC UGCUAAAUGC GCAAGCAUGG +156/157/158 CATGCTTGCAT CAUGCUUGC CAUUUAGCA 0114 ise2c.pk001.i23translation AAGATCAGAAGA GAUCAGAAGA UCCGGCCAAU + 159/160/161 initiationTTGGCCGGAAG UUGGCCGGA CUUCUGAUC factor 0115 AATTCTTCAGCA UUCUUCAGCAGUAUCGAUUU NT 162/163/164 AATCGATACCA AAUCGAUAC GCUGAAGAA 0116AAATGCTGTCAA AUGCUGUCAA UAAAUCCUCU 165/166/167 GAGGATTTAAA GAGGAUUUAUGACAGCAU 0117 ise2c.pk001.l24 translation AAGCTCGAGACT GCUCGAGACUUCAAGAGCAA + 168/169/170 initiation TGCTCTTGATG UGCUCUUGA GUCUCGAGCfactor 0118 AACTGTTAGCTC CUGUUAGCUC GCAGACCUUG + 171/172/173 AAGGTCTGCTAAAGGUCUGC AGCUAACAG 0119 AAGACTTTCTAT GACUUUCUAU CAAAUUCUGA 174/175/176CAGAATTTGCG CAGAAUUUG UAGAAAGUC 0120 ise2c.pk005.b9 translationAAACTTAATCAT ACUUAAUCAU UCGUCGUCCA 177/178/179 initiation GGACGACGACAGGACGACGA UGAUUAAGU factor 0121 AAAGAAGAAGAA AGAAGAAGAA CCCUUCUUCU +180/181/182 GAAGAAGGGAG GAAGAAGGG UCUUCUUCU 0122 AAGATCAAGAGA GAUCAAGAGACCUCGACAUU + 183/184/185 ATGTCGAGGAT AUGUCGAGG CUCUUGAUC 0123ise2c.pk002.m10 SAR1 AAAATCGTCGGT AAUCGUCGGU GUCGCUAAAA + 186/187/188TTTAGCGACGT UUUAGCGAC CCGACGAUU 0124 AACTGTCAATAG CUGUCAAUAGGCAUACUGCC + 189/190/191 GCAGTATGCGT GCAGUAUGC UAUUGACAG 0125AACCTGTACCAA CCUGUACCAA AGUGGUCUGU + 192/193/194 CAGACCACTGG CAGACCACUUGGUACAGG 0126 ise2c.pk001.c14 Elongation AACCAAAAATGG CCAAAAAUGGUUUCCUUGCC + 195/196/197 factor GCAAGGAAAAG GCAAGGAAA CAUUUUUGG 0127AACGTGGTATCA CGUGGUAUCA UAUCGAUGGU + 198/199/200 CCATCGATATT CCAUCGAUAGAUACCACG 0128 AACAAAATGGAC CAAAAUGGAC CUCAGUGGAG + 201/202/203TCCACTGAGCC UCCACUGAG UCCAUUUUG 0129 ise2c.pk001.d16 ElongationAATCCGTGACTA UCCGUGACUA AUUUUUGGUU + 204/205/206 factor ACCAAAAATGGACCAAAAAU AGUCACGGA 0130 AACATTGTCGTC CAUUGUCGUC GUGUCCAAUG +207/208/209 ATTGGACACGT AUUGGACAC ACGACAAUG 0131 ise2c.pk005.h3phosphooligo- AATTTGTGAGAC UUUGUGAGAC CGGCCACCAG 421/422/423 saccharide TGGTGGCCGAA UGGUGGCCG UCUCACAAA ... 0132 AATCTGATTGTA UCUGAUUGUAGGGGGCGAAU 424/425/426 TTCGCCCCCTC UUCGCCCCC ACAAUCAGA 0133 AACACTCTAGTTCACUCUAGUU AAUAGGCAGA 427/428/429 CTGCCTATTCT CUGCCUAUU ACUAGAGUG 0134ise2c.pk001.d21 myosin AACACACATCAC CACACAUCAC UCCGCCAUUG 430/431/432AATGGCGGATA AAUGGCGGA UGAUGUGUG 0135 AAGGATGGCATC GGAUGGCAUC CUUGCCGAUG433/434/435 ATCGGCAAGAA AUCGGCAAG AUGCCAUCC 0136 AAAGGCTTCATC AGGCUUCAUCCGCGGUGUCG 436/437/438 GACACCGCGAA GACACCGCG AUGAAGCCU 0137ise2c.pk001.j9 myosin AAACTCCAATTA ACUCCAAUUA AGUAGGUUAU 210/211/212TAACCTACTAG UAACCUACU AAUUGGAGU 0138 AAGTACAAGGAT GUACAAGGAU GCCGAUCAGA213/214/215 CTGATCGGCAA CUGAUCGGC UCCUUGUAC 0139 AAGACTTTCTTC GACUUUCUUCGGGCCACAUG 216/217/218 ATGTGGCCCAT AUGUGGCCC AAGAAAGUC 0140ise2c.pk002.f12 myosin AAACAAAGTATC ACAAAGUAUC GGUGUAGGCG 439/440/441GCCTACACCGC GCCUACACC AUACUUUGU 0141 AATAGCGTCGAT UAGCGUCGAU UCGUUGAAGA442/443/444 CTTCAACGACT CUUCAACGA UCGACGCUA 0142 ise2c.pk001.b14potassium AACTCATAGAGC CUCAUAGAGC ACACAUCAAG 219/220/221 channelTTGATGTGTGG UUGAUGUGU CUCUAUGAG amino acid transporter 0143 AAGATGTGGATGGAUGUGGAUG CAGUGACGUC + 221/223/224 ACGTCACTGGT ACGUCACUG AUCCACAUC 0144AACCTTCCTGAT CCUUCCUGAU CAGAAGAGAA + 225/226/227 TCTCTTCTGTG UCUCUUCUGUCAGGAAGG 0145 ise2c.pk003.f2 potassium AACAGTGCTTGT CAGUGCUUGUUCACUUAUCA + 228/229/230 inwardly GATAAGTGAAC GAUAAGUGA CAAGCACUGrectifier  . . . 0146 AAGTTAATGGTG GUUAAUGGUG GAGGGCAGUC + 231/232/233ACTGCCCTCGA ACUGCCCUC ACCAUUAAC 0147 AATAAAGCGATG UAAAGCGAUGCUAUGGGGUC + 234/235/236 ACCCCATAGGA ACCCCAUAG AUCGCUUUA 0148ise2c.pk005.l20 amino acid AAACGGTACTGC ACGGUACUGC CUUUUUGCUG +237/238/239 transporter AGCAAAAAGAC AGCAAAAAG CAGUACCGU 0149AAGCTGCATACT GCUGCAUACU GAGCCAAGAA + 240/241/242 TCTTGGCTCTC UCUUGGCUCGUAUGCAGC 0150 AAATGTTTACAG AUGUUUACAG AUCGCGUCUC + 243/244/245AGACGCGATGA AGACGCGAU UGUAAACAU 0151 ise2c.pk001.d1 tubulin AACGTCGATCTTCGUCGAUCUU GAACUCGGUA + 246/247/248 ACCGAGTTCCA ACCGAGUUC AGAUCGACG 0152ise2c.pk001.k6 tubulin AATTCAAAATGC UUCAAAAUGC UGCACUCACG 249/250/251GTGAGTGCATC GUGAGUGCA CAUUUUGAA 0153 AAATCGTAGACC AUCGUAGACCCGAGGACUAG + 252/253/254 TAGTCCTCGAC UAGUCCUCG GUCUACGAU 0154ise2c.pk001.l2 tubulin AAACTCAATTCA ACUCAAUUCA CACGCAUUUU + 255/256/257AAATGCGTGAG AAAUGCGUG GAAUUGAGU 0155 AACTTATCACTG CUUAUCACUG CUUCCUUACC258/259/260 GTAAGGAAGAT GUAAGGAAG AGUGAUAAG 0156 ise2c.pk002.b4ubiquitin AAGAGTTACGAA GAGUUACGAA UGGUGACGGU + 261/262/263 CCGTCACCATACCGUCACCA UCGUAACUC 0157 AAACTTAGTCCG ACUUAGUCCG UUCAUUAUCC +264/265/266 GATAATGAACC GAUAAUGAAt GGACUAAGU 0158 AAGGCGATGTACGGCGAUGUAC CAGGUUCUCG + 267/268/269 GAGAACCTGTT GAGAACCUG UACAUCGCC 0159ise2c.pk001.j16 small AACGACAAGATG CGACAAGAUG CUCCUUCAGC + 270/271/272nuclear CTGAAGGAGAC CUGAAGGAG AUCUUGUCG ribonucleo- protein 0160ise2c.pk006.h23  small AAGATAAAGGTC GAUAAAGGUC UCCACACGCG + nuclearGCGTGTGGACC GCGUGUGGA ACCUUUAUC ribonucleo-  protein 0161 AATGTCAAGACTUGUCAAGACU GUUUGGAUCA + 276/277/278 GATCCAAACAC GAUCCAAAC GUCUUGACA 0162AACATTCGAGTC CAUUCGAGUC ACCUGUUCAG + 279/280/281 TGAACAGGTGG UGAACAGGUtACUCGAAUG (Note: the sense RNA primer sequence and the antisense RNAprimer sequences shown in table 8 were generated with 2 thymine residuesat the 3′ end.)

TABLE 9 SEQ ID NO Target region/ 30 15 8 sense/ Sample seq id gene idTarget sequence forward reverse ppm ppm ppm antisense 0075AAGGTCGCTGACGA GGUCGCUGACG CUUGUUCUCGUCA 51/52/53 GAACAAGGA AGAACAAGGCGACC 0076 AAGTGTCCTGGGCTT GUGUCCUGGGC GAACUCAAGCCCA + 54/55/56GAGTTCCA UUGAGUUC GGACAC 0077 ise1c.pk003.f7 Juvenile AAGAAGAAGCTCCTGAAGAAGCUCC CACGUGGAGGAGC 57/58/59 hormone CCACGTGTT UCCACGUG UUCUUCquery 0078 AAGGTCGCTGACGA GGUCGCUGACG CUUGUUCUCGUCA 60/61/62 GAACAAGGAAGAACAAG GCGACC 0079 AATGTCCTGGGGCT UGUCCUGGGGC GAAACUCAGCCCC 63/64/65GAGTTTCAA UGAGUUUC AGGACA 0080 ise1c.pk005.a15 Juvenile AAGAATAAGCTCCTGAAUAAGCUCC CACGUGGAGGAGC + + 66/67/68 hormone  CCACGTGTT UCCACGUGUUAUUC query 0081 AATTTGTCGAGGAG UUUGUCGAGGA AUAGGGUCUCCUC 69/70/71ACCCTATTG GACCCUAU GACAAA 0082 AAGTTCGCGTTCACT GUUCGCGUUCA UUCAAGAGUGAANT 72/73/74 CTTGAAGA CUCUUGAA CGCGAAC 0083 ise1c.pk006.d24 JuvenileAACTGCCCCTTAACC CUGCCCCUUAAC AGAUGAGGUUAA 75/76/77 hormone  TCATCTATCUCAUCUtt GGGGCAG query 0084 AATCACGCTGAAAC UCACGCUGAAA UACAGUGGUUUC +78/79/80 CACTGTATA CCACUGUAtt AGCGUGA 0085 ise2c.pk009.i4 JuvenileAAAATATGGCGCGC AAUAUGGCGCG ACAAUAGGCGCGC 81/82/83 hormone  CTATTGTTTCCUAUUGU CAUAUU query 0086 AACGTTCTCGGTCTT CGUUCUCGGUC CAGUGAAAGACCG84/85/86 TCACTGCT UUUCACUG AGAACG 0087 AAGTCATCGTTCCAA GUCAUCGUUCCGUAGACUUGGAA + 87/88/89 GTCTACCT AAGUCUAC CGAUGAC 0088 ise2c.pk001.d19vacuolar AACCCCTTGAATGTT CCCCUUGAAUG GACCUUAACAUUC + 90/91/92 query AAGGTCGG UUAAGGUC AAGGGG 0089 AAGTACACCATGTT GUACACCAUGU UACUUGCAACAUG93/94/95 GCAAGTATG UGCAAGUA GUGUAC 0090 AACGTGTCCATGAT CGUGUCCAUGAGUCAGCCAUCAUG NT 96/97/98 GGCTGACTC UGGCUGAC GACACG 0091 ise2c.pk001.e14vacuolar AAACCTACAAAATG ACCUACAAAAU UUUCGGCCAUUUU  99/100/101 query GCCGAAAAC GGCCGAAA GUAGGU 0092 AATCTACGGACCCTT UCUACGGACCCCCAAAGAAGGGUC + 102/103/104 CTTTGGAG UUCUUUGG CGUAGA 0093ise2c.pk001.f20 vacuolar AACTCTGACGTCATC CUCUGACGUCA GUAGAUGAUGAC105/106/107 query  ATCTACGT UCAUCUAC GUCAGAG 0094 AAGTGCTTGGGTAAGUGCUUGGGUA GUCGGGGUUACCC 108/109/110 CCCCGACAG ACCCCGAC AAGCAC 0095AACTGGCTCATCTCC CUGGCUCAUCU GCUGUAGGAGAU 111/112/113 TACAGCAA CCUACAGCGAGCCAG 0096 ise2c.pk010.h3 cadherin AAACAGTGCGTCGT ACAGUGCGUCGAUAUAUUACGAC 114/115/116 query  AATATATTC UAAUAUAU GCACUGU 0097AAGGCACATGGTCC GGCACAUGGUC CAGUGAAGGACCA 117/118/119 TTCACTGAT CUUCACUGUGUGCC 0098 AACACCATGACCCT CACCAUGACCCU GUACACGAGGGUC NT 120/121/122CGTGTACAA CGUGUAC AUGGUG 0099 ise2c.pk007.k24 cuticle AACGAGGCCGGATCCGAGGCCGGAU CUUAAGAGAUCCG 123/124/125 protein TCTTAAGCA CUCUUAAG GCCUCG0100 AACTTCACACATAA CUUCACACAUA UGUCUAGUUAUG 457/458/459 CTAGACAAAACUAGACA UGUGAAG 0101 AATGCGTGGCGATTT UUAGAAAUUAU CUGGGCUUAUAA460/461/462 CAAACTTA AAGCCCAG UUUCUAA 0102 ise2c.pk011.a10 cuticle AAAAAACACAGACC AAAACACAGAC UGAACGUGGUCU 463/464/465 protein ACGTTCACACACGUUCA GUGUUUU 0103 AATCGATGGTGGTG UCGAUGGUGGU CGAAUAACACCAC +126/127/128 TTATTCGCT GUUAUUCG CAUCGA 0104 ise2c.pk011.h12 cuticle AAAGAAAATGCTAC AGAAAAUGCUA GUAACGCGUAGCA 129/130/131 protein GCGTTACGACGCGUUAC UUUUCU 0105 AACCCTTGGACACT CCCUUGGACAC UUCCAGUAGUGUC +132/133/134 ACTGGAAGA UACUGGAA CAAGGG 0106 AAGGATCCTATGTGT GGAUCCUAUGUCCUGGUACACAUA NT 135/136/137 ACCAGGTT GUACCAGG GGAUCC 0107ise2c.pk001.d22 translation AAACTCGGCACACA ACUCGGCACAC AUUGUGUUGUGU138/139/140 initiation  ACACAATGG AACACAAU GCCGAGU factor 0108AATACGAAGATATC UACGAAGAUAU AAGGGCAGAUAU + 141/142/143 TGCCCTTCC CUGCCCUUCUUCGUA 0109 AATCAACAGCTCTTA UCAACAGCUCU UUUAUGUAAGAG + 144/145/146CATAAATG UACAUAAA CUGUUGA 0110 ise2c.pk001.d9 translation AAAGAAGATCAGAAAGAAGAUCAGA GCCAAUCUUCUGA 147/148/149 initiation  GATTGGCCG AGAUUGGCUCUUCU factor 0111 AAAAGCCGTCTGCT AAGCCGUCUGC GUUGGAUAGCAG 150/151/152ATCCAACAA UAUCCAAC ACGGCUU 0112 AATGCTAAATGCCA UGCUAAAUGCCGCAAGCAUGGCAU + 153/154/155 TGCTTGCAT AUGCUUGC UUAGCA 0113ise2c.pk001.i23 translation AAGATCAGAAGATT GAUCAGAAGAU UCCGGCCAAUCUU +156/157/158 initiation  GGCCGGAAG UGGCCGGA CUGAUC factor 0114AATTCTTCAGCAAAT UUCUUCAGCAA GUAUCGAUUUGC NT 159/160/161 CGATACCAAUCGAUAC UGAAGAA 0115 AAATGCTGTCAAGA AUGCUGUCAAG UAAAUCCUCUUGA +162/163/164 GGATTTAAA AGGAUUUA CAGCAU 0116 ise2c.pk001.i24 translationAAGCTCGAGACTTG GCUCGAGACUU UCAAGAGCAAGUC + 165/166/167 initiation CTCTTGATG GCUCUUGA UCGAGC factor 0117 AACTGTTAGCTCAA CUGUUAGCUCAGCAGACCUUGAGC + 168/169/170 GGTCTGCTA AGGUCUGC UAACAG 0118AAGACTTTCTATCAG GACUUUCUAUC CAAAUUCUGAUA 171/172/173 AATTTGCG AGAAUUUGGAAAGUC 0119 ise2c.pk005.b9 translation AAACTTAATCATGG ACUUAAUCAUGUCGUCGUCCAUGA 174/175/176 initiation  ACGACGACA GACGACGA UUAAGU factor0120 AAAGAAGAAGAAGA AGAAGAAGAAG CCCUUCUUCUUCU + 177/178/179 AGAAGGGAGAAGAAGGG UCUUCU 0121 AAGATCAAGAGAAT GAUCAAGAGAA CCUCGACAUUCUC + + +180/181/182 GTCGAGGAT UGUCGAGG UUGAUC 0122 AAAATCGTCGGTTTT AAUCGUCGGUUGUCGCUAAAACCG NT 183/184/185 AGCGACGT UUAGCGAC ACGAUU 0123ise2c.pk002.m10 SAR1 AACTGTCAATAGGC CUGUCAAUAGG GCAUACUGCCUAU +186/187/188 AGTATGCGT CAGUAUGC UGACAG 0124 AACCTGTACCAACA CCUGUACCAACAGUGGUCUGUUG + 189/190/191 GACCACTGG AGACCACU GUACAGG 0125ise2c.pk001.c14 Elongation AACCAAAAATGGGC CCAAAAAUGGG UUUCCUUGCCCAU +192/193/194 factor AAGGAAAAG CAAGGAAA UUUUGG 0126 AACGTGGTATCACCCGUGGUAUCAC UAUCGAUGGUGA + 195/196/197 ATCGATATT CAUCGAUA UACCACG 0127AACAAAATGGACTC CAAAAUGGACU CUCAGUGGAGUCC + 198/199/200 CACTGAGCCCCACUGAG AUUUUG 0128 ise2c.pk001.d16 Elongation AATCCGTGACTAACUCCGUGACUAA AUUUUUGGUUAG + 201/202/203 factor CAAAAATGG CCAAAAAU UCACGGA0129 AACATTGTCGTCATT CAUUGUCGUCA GUGUCCAAUGACG 204/205/206 GGACACGTUUGGACAC ACAAUG 0130 ise2c.pk005.h3 phosphooligo- AATTTGTGAGACTGUUUGUGAGACU CGGCCACCAGUCU NT 207/208/209 saccharide GTGGCCGAA GGUGGCCGCACAAA . . . 0131 AATCTGATTGTATTC UCUGAUUGUAU GGGGGCGAAUAC 421/422/423GCCCCCTC UCGCCCCC AAUCAGA 0132 AACACTCTAGTTCTG CACUCUAGUUC AAUAGGCAGAAC424/425/426 CCTATTCT UGCCUAUU UAGAGUG 0133 ise2c.pk001.d21 myosinAACACACATCACAA CACACAUCACA UCCGCCAUUGUGA 427/428/429 TGGCGGATA AUGGCGGAUGUGUG 0134 AAGGATGGCATCAT GGAUGGCAUCA CUUGCCGAUGAUG 430/431/432CGGCAAGAA UCGGCAAG CCAUCC 0135 AAAGGCTTCATCGA AGGCUUCAUCG CGCGGUGUCGAUG433/434/435 CACCGCGAA ACACCGCG AAGCCU 0136 ise2c.pk001.j9 myosinAAACTCCAATTATA ACUCCAAUUAU AGUAGGUUAUAA 436/437/438 ACCTACTAG AACCUACUUUGGAGU 0137 AAGTACAAGGATCT GUACAAGGAUC GCCGAUCAGAUCC 210/211/212GATCGGCAA UGAUCGGC UUGUAC 0138 AAGACTTTCTTCATG GACUUUCUUCA GGGCCACAUGAAGNT 213/214/215 TGGCCCAT UGUGGCCC AAAGUC 0139 ise2c.pk002.f12 myosinAAACAAAGTATCGC ACAAAGUAUCG GGUGUAGGCGAU 216/217/218 CTACACCGC CCUACACCACUUUGU 0140 AATAGCGTCGATCTT UAGCGUCGAUC UCGUUGAAGAUC 439/440/441CAACGACT UUCAACGA GACGCUA 0141 ise2c.pk001.b14 potassium AACTCATAGAGCTTCUCAUAGAGCU ACACAUCAAGCUC 442/443/444 channel  GATGTGTGG UGAUGUGU UAUGAGamino acid  transporter 0142 AAGATGTGGATGAC GAUGUGGAUGA CAGUGACGUCAUC219/220/221 GTCACTGGT CGUCACUG CACAUC 0143 AACCTTCCTGATTCT CCUUCCUGAUUCAGAAGAGAAUC + 222/223/224 CTTCTGTG CUCUUCUG AGGAAGG 0144 ise2c.pk003.f2potassium AACAGTGCTTGTGAT CAGUGCUUGUG UCACUUAUCACAA + + 225/226/227inwardly AAGTGAAC AUAAGUGA GCACUG rectifier . . . 0145 AAGTTAATGGTGACGUUAAUGGUGA GAGGGCAGUCACC + 228/229/230 TGCCCTCGA CUGCCCUC AUUAAC 0146AATAAAGCGATGAC UAAAGCGAUGA CUAUGGGGUCAUC NT 231/232/233 CCCATAGGACCCCAUAG GCUUUA 0147 ise2c.pk005.l20 amino acid AAACGGTACTGCAGACGGUACUGCA CUUUUUGCUGCAG + 234/235/236 transporter CAAAAAGAC GCAAAAAGUACCGU 0148 AAGCTGCATACTTCT GCUGCAUACUU GAGCCAAGAAGU 237/238/239TGGCTCTC CUUGGCUC AUGCAGC 0149 AAATGTTTACAGAG AUGUUUACAGAAUCGCGUCUCUGU + 240/241/242 ACGCGATGA GACGCGAU AAACAU 0150ise2c.pk001.d1 tubulin AACGTCGATCTTACC CGUCGAUCUUA GAACUCGGUAAG + +243/244/245 GAGTTCCA CCGAGUUC AUCGACG 0151 ise2c.pk001.k6 tubulinAATTCAAAATGCGT UUCAAAAUGCG UGCACUCACGCAU + 246/247/248 GAGTGCATCUGAGUGCA UUUGAA 0152 AAATCGTAGACCTA AUCGUAGACCU CGAGGACUAGGUC + +249/250/251 GTCCTCGAC AGUCCUCG UACGAU 0153 ise2c.pk001.l2 tubulinAAACTCAATTCAAA ACUCAAUUCAA CACGCAUUUUGAA + + 252/253/254 ATGCGTGAGAAUGCGUG UUGAGU 0154 AACTTATCACTGGTA CUUAUCACUGG CUUCCUUACCAGU NT255/256/257 AGGAAGAT UAAGGAAG GAUAAG 0155 ise2c.pk002.b4 ubiquitinAAGAGTTACGAACC GAGUUACGAAC UGGUGACGGUUC + 258/259/260 GTCACCATA CGUCACCAGUAACUC 0156 AAACTTAGTCCGGA ACUUAGUCCGG UUCAUUAUCCGGA + + 261/262/263TAATGAACC AUAAUGAA CUAAGU 0157 AAGGCGATGTACGA GGCGAUGUACGCAGGUUCUCGUAC + 264/265/266 GAACCTGTT AGAACCUG AUCGCC 0158ise2c.pk001.j16 small  AACGACAAGATGCT CGACAAGAUGC CUCCUUCAGCAUC +267/268/269 nuclear GAAGGAGAC UGAAGGAG UUGUCG ribonucleo- protein 0159ise2c.pk006.h23 small  AAGATAAAGGTCGC GAUAAAGGUCG UCCACACGCGACC +270/271/272 nuclear GTGTGGACC CGUGUGGA UUUAUC ribonucleo- protein 0160AATGTCAAGACTGA UGUCAAGACUG GUUUGGAUCAGU + 273/274/275 TCCAAACAC AUCCAAACCUUGACA 0161 AACATTCGAGTCTG CAUUCGAGUCU ACCUGUUCAGACU + + 276/277/278AACAGGTGG GAACAGGU CGAAUG (Note: the sense RNA primer sequence and theantisense RNA primer sequences shown in table 9 were generated with 2thymine residuesat the 3′ end.)

TABLE 10 Injection Droplet Topical Topical Topical Topical TopicalTopical Mortality Feeding assay 1 Assay 2 Assay 3 Assay 4 Assay 4 Assay4 Top. 5 Top. 5 well seq i.d. midgut gene id (%) Result 30 ppm 30 ppm 30ppm 30 ppm 15 ppm 8 ppm 15 ppm 8 ppm 77 ise1c.pk002.m13 no Juvenile NTNT NT − − + + hormone query 81 ise1c.pk005.a15 no Juvenile NT NT NT −− + + + hormone query 114 ise2c.pk001.i23 no translation NT NTNT + + + + initiation factor 122 ise2c.pk005.b9 Yes translation NT NTNT + + + + + + + initiation factor 143 ise2c.pk001.b14 no potassium NTNT NT − + + channel ami- acid transporter 145 ise2c.pk003.f2 Yespotassium NT NT NT + + + + + inwardly rectifier . . . 146 ise2c.pk003.f2Yes potassium NT NT NT + + + + inwardly rectifier . . . 149ise2c.pk005.l20 Yes hypothetical NT NT NT + + + sodium dependenttransport 151 ise2c.pk001.d1 No alpha tubulin NT NT NT + + + + + 154ise2c.pk001.l2 No alpha tubulin NT NT NT + + + + + 157 ise2c.pk002.b4Yes Probable NT NT NT + + + + + ubiquitin-- protein ligase 158ise2c.pk002.b4 Yes Probable NT NT NT + + − + ubiquitin-- protein ligase162 ise2c.pk006.h23 Yes RNA- NT NT NT + + + + + binding protein squid

TABLE 11 DsRNA # gene id seqID Comment 8 ppm 4 ppm 2 ppm 0163pre-mRNA-binding protein ise2c.pk006.m8 Plate 2 A1 0164 pre-mRNA-bindingprotein ise2c.pk006.m8 Plate 2 B1 0165 pre-mRNA-binding proteinise2c.pk006.m8 Plate 2 C1 0166 pre-mRNA-binding protein ise2c.pk006.m8Plate 2 D1 S 0167 pre-mRNA-binding protein ise2c.pk006.m8 Plate 2 E1 S0168 chymotrypsin-like; protease ise2c.pk001.a23 Plate 2 F1 S 0169chymotrypsin-like; protease ise2c.pk001.a23 Plate 2 G1 0170chymotrypsin-like; protease ise2c.pk001.a23 Plate 2 H1 0171chymotrypsin-like; protease ise2c.pk001.a23 Plate 2 A2 S 0172chymotrypsin-like; protease ise2c.pk001.a23 Plate 2 B2 S 0173chymotrypsinogen; protease ise2c.pk001.a7 Plate 2 C2 S 0174chymotrypsinogen; protease ise2c.pk001.a7 Plate 2 D2 0175chymotrypsinogen; protease ise2c.pk001.a7 Plate 2 E2 0176chymotrypsinogen; protease ise2c.pk001.a7 Plate 2 F2 0177chymotrypsinogen; protease ise2c.pk001.a7 Plate 2 G2 0178actin-depolymerizing ise2c.pk004.c4 Plate 2 H2 0179 actin-depolymerizingise2c.pk004.c4 Plate 2 A3 0180 actin-depolymerizing ise2c.pk004.c4 Plate2 B3 0181 actin-depolymerizing ise2c.pk004.c4 Plate 2 C3 0182actin-depolymerizing ise2c.pk004.c4 Plate 2 D3 0183 actin depolymerizingfactor ise2c.pk004.l4 Plate 2 E3 0184 actin depolymerizing factorise2c.pk004.l4 Plate 2 F3 0185 actin depolymerizing factorise2c.pk004.l4 Plate 2 G3 0186 actin depolymerizing factorise2c.pk004.l4 Plate 2 H3 0187 actin depolymerizing factorise2c.pk004.l4 Plate 2 A4 0188 dismutase; superoxide ise2c.pk004.n19Plate 2 B4 0189 dismutase; superoxide ise2c.pk004.n19 Plate 2 C4 0190dismutase; superoxide ise2c.pk004.n19 Plate 2 D4 0191 dismutase;superoxide ise2c.pk004.n19 Plate 2 E4 0192 dismutase; superoxideise2c.pk004.n19 Plate 2 F4 0193 superoxide dismutase ise2c.pk005.f21Plate 2 G4 0194 superoxide dismutase ise2c.pk005.f21 Plate 2 H4 0195superoxide dismutase ise2c.pk005.f21 Plate 2 A5 0196 adenylate kinaseisozyme 3 ise2c.pk010.h5 Plate 2 B5 0197 adenylate kinase isozyme 3ise2c.pk010.h5 Plate 2 C5 0198 adenylate kinase isozyme 3 ise2c.pk010.h5Plate 2 D5 0199 adenylate kinase isozyme 3 ise2c.pk010.h5 Plate 2 E50200 adenylate kinase isozyme 3 ise2c.pk010.h5 Plate 2 F5 0201 ecdysoneoxidase ise2c.pk001.c18 Plate 2 G5 0202 ecdysone oxidase ise2c.pk001.c18Plate 2 H5 0203 ecdysone oxidase ise2c.pk001.c18 Plate 2 A6 0204innexin-2 ise2c.pk004.p1 Plate 2 B6 0205 innexin-2 ise2c.pk004.p1 Plate2 C6 0206 innexin-2 ise2c.pk004.p1 Plate 2 D6 0207 innexin-2ise2c.pk004.p1 Plate 2 E6 0208 innexin-2 ise2c.pk004.p1 Plate 2 F6

TABLE 12 DsRNA# gene id seqID Comment 16 ppm 8 ppm 4 ppm 0163pre-mRNA-binding protein ise2c.pk006.m8 Plate 2 A1 S 0164pre-mRNA-binding protein ise2c.pk006.m8 Plate 2 B1 S 0165pre-mRNA-binding protein ise2c.pk006.m8 Plate 2 C1 S 0166pre-mRNA-binding protein ise2c.pk006.m8 Plate 2 D1 S 0167pre-mRNA-binding protein ise2c.pk006.m8 Plate 2 E1 S 0168chymotrypsin-like; protease ise2c.pk001.a23 Plate 2 F1 ss S 0169chymotrypsin-like; protease ise2c.pk001.a23 Plate 2 G1 ss 0170chymotrypsin-like; protease ise2c.pk001.a23 Plate 2 H1 0171chymotrypsin-like; protease ise2c.pk001.a23 Plate 2 A2 0172chymotrypsin-like; protease ise2c.pk001.a23 Plate 2 B2 0173chymotrypsinogen; protease ise2c.pk001.a7 Plate 2 C2 ss 0174chymotrypsinogen; protease ise2c.pk001.a7 Plate 2 D2 S 0175chymotrypsinogen; protease ise2c.pk001.a7 Plate 2 E2 S 0176chymotrypsinogen; protease ise2c.pk001.a7 Plate 2 F2 S 0177chymotrypsinogen; protease ise2c.pk001.a7 Plate 2 G2 S 0178actin-depolymerizing ise2c.pk004.c4 Plate 2 H2 0179 actin-depolymerizingise2c.pk004.c4 Plate 2 A3 ss 0180 actin-depolymerizing ise2c.pk004.c4Plate 2 B3 s 0181 actin-depolymerizing ise2c.pk004.c4 Plate 2 C3 0182actin-depolymerizing ise2c.pk004.c4 Plate 2 D3 0183 actin depolymerizingfactor ise2c.pk004.l4 Plate 2 E3 0184 actin depolymerizing factorise2c.pk004.l4 Plate 2 F3 0185 actin depolymerizing factorise2c.pk004.l4 Plate 2 G3 0186 actin depolymerizing factorise2c.pk004.l4 Plate 2 H3 0187 actin depolymerizing factorise2c.pk004.l4 Plate 2 A4 0188 dismutase; superoxide ise2c.pk004.n19Plate 2 B4 0189 dismutase; superoxide ise2c.pk004.n19 Plate 2 C4 s 0190dismutase; superoxide ise2c.pk004.n19 Plate 2 D4 0191 dismutase;superoxide ise2c.pk004.n19 Plate 2 E4 0192 dismutase; superoxideise2c.pk004.n19 Plate 2 F4 s 0193 superoxide dismutase ise2c.pk005.f21Plate 2 G4 0194 superoxide dismutase ise2c.pk005.f21 Plate 2 H4 0195superoxide dismutase ise2c.pk005.f21 Plate 2 A5 0196 adenylate kinaseisozyme 3 ise2c.pk010.h5 Plate 2 B5 0197 adenylate kinase isozyme 3ise2c.pk010.h5 Plate 2 C5 0198 adenylate kinase isozyme 3 ise2c.pk010.h5Plate 2 D5 0199 adenylate kinase isozyme 3 ise2c.pk010.h5 Plate 2 E50200 adenylate kinase isozyme 3 ise2c.pk010.h5 Plate 2 F5 0201 ecdysoneoxidase ise2c.pk001.c18 Plate 2 G5 0202 ecdysone oxidase ise2c.pk001.c18Plate 2 H5 0203 ecdysone oxidase ise2c.pk001.c18 Plate 2 A6 0204innexin-2 ise2c.pk004.p1 Plate 2 B6 0205 innexin-2 ise2c.pk004.p1 Plate2 C6 0206 innexin-2 ise2c.pk004.p1 Plate 2 D6 0207 innexin-2ise2c.pk004.p1 Plate 2 E6 0208 innexin-2 ise2c.pk004.p1 Plate 2 F6

TABLE 13 Summary of FAW droplet feeding data for the first set ofsynthetic dsRNA primers Assay #4 Assay #1 Assay #2 Assay #3 (table 8)(table 6) (table 7) (table 8) 30 15 8 30 ppm 30 ppm 30 ppm ppm ppm ppm0075 ise1c.pk002.m13 Juvenile hormone query − − − NT − − 0076 NT − − − −− 0077 NT − − + − − 0078 ise1c.pk003.f7 Juvenile hormone query NT − − −− − 0079 NT − − − − − 0080 NT − + − − − 0081 ise1c.pk005.a15 Juvenilehormone query NT − − + + − 0082 NT − − − − − 0083 + + NT NT − − 0084ise1c.pk006.d24 Juvenile hormone query NT − + − − − 0085 + − − + − −0086 ise2c.pk009.i4 Juvenile hormone query − − − − − − 0087 NT − − − − −0088 + − − + − − 0089 ise2c.pk001.d19 vacuolar query + − + + − − 0090 NT− − − − − 0091 + − + NT − − 0092 ise2c.pk001.e14 vacuolar query NT − − −− − 0093 NT − − + − − 0094 ise2c.pk001.f20 vacuolar query + − − − − −0095 + − − − − − 0096 NT − − − − − 0097 ise2c.pk010.h3 cadherin query NT− − − − − 0098 NT + − − − − 0099 NT − − NT − − 0100 ise2c.pk007.k24cuticle protein NT − − − − − 0101 NT − − − − − 0102 NT − − − − − 0103ise2c.pk011.a10 cuticle protein NT + − − − − 0104 NT + + + − − 0105ise2c.pk011.h12 cuticle protein NT − − − − − 0106 NT + − + − − 0107 + −− NT − − 0108 ise2c.pk001.d22 translation initiation factor NT − + − − −0109 NT + + + − − 0110 NT − + + − − 0111 ise2c.pk001.d9 translationinitiation factor NT − − − − − 0112 NT + − − − − 0113 NT − + + − − 0114ise2c.pk001.i23 translation initiation factor NT + + + − − 0115 − − NTNT − − 0116 NT − − + − − 0117 ise2c.pk001.l24 translation initiationfactor NT − + + − − 0118 NT − + + − − 0119 NT + − − − − 0120ise2c.pk005.b9 translation initiation factor NT − − − − − 0121 NT + + +− − 0122 NT + + + + + 0123 ise2c.pk002.m10 SAR1 − − + NT − − 0124 NT− + + − − 0125 NT + + + − − 0126 ise2c.pk001.c14 Elongation factorNT + + + − − 0127 NT + + + − − 0128 NT − + + − − 0129 ise2c.pk001.d16Elongation factor NT + + + − − 0130 NT − + − − − 0131 ise2c.pk005.h3phosphooligosaccharide . . . − − − NT − − 0132 NT − − − − − 0133 NT − −− − − 0134 ise2c.pk001.d21 myosin NT − − − − − 0135 NT − − − − − 0136 NT− − − − − 0137 ise2c.pk001.j9 myosin NT − − − − − 0138 NT + − − − − 0139− − − NT − − 0140 ise2c.pk002.f12 myosin NT − − − − − 0141 NT − − − − −0142 ise2c.pk001.b14 potassium channel amino acid NT − − − − −transporter 0143 NT − + − − − 0144 NT − + + − − 0145 ise2c.pk003.f2potassium inwardly rectifier . . . NT + + + + − 0146 NT + + + − −0147 + + + NT − − 0148 ise2c.pk005.l20 amino acid transporter NT + + + −− 0149 NT + + − − − 0150 NT − + + − − 0151 ise2c.pk001.d1 tubulinNT + + + + − 0152 ise2c.pk001.k6 tubulin NT − − + − − 0153 NT + + + + −0154 ise2c.pk001.l2 tubulin NT + + + + − 0155 − − − NT − − 0156ise2c.pk002.b4 ubiquitin NT − + + − − 0157 NT + + + + − 0158 NT + + + −− 0159 ise2c.pk001.j16 small nuclear ribonucleoprotein NT + + + − − 0160ise2c.pk006.h23 small nuclear ribonucleoprotein NT − + + − − 0161 NT− + + − − 0162 NT + + + + −

Example 2 Transformation of Maize

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing the silencing element of the invention operablylinked to either a tissue specific, tissue selective, or constitutivepromoter and the selectable marker gene PAT (Wohlleben et al. (1988)Gene 70:25-37), which confers resistance to the herbicide Bialaphos. Inone embodiment, the constructs will have 2 identical 2-300 bp segmentsof the target gene in opposite orientations with an “intron” segmentbetween them acting as a hairpin loop. Such a construct can be linked tothe dMMB promoter. Alternatively, the selectable marker gene is providedon a separate plasmid. Transformation is performed as follows. Mediarecipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% Clorox bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5 cm target zone in preparation forbombardment.

A plasmid vector comprising the silencing element of interest operablylinked to either the tissue specific, tissue selective, or constitutivepromoter is made. This plasmid DNA plus plasmid DNA containing a PATselectable marker is precipitated onto 1.1 μm (average diameter)tungsten pellets using a CaCl₂ precipitation procedure as follows: 100μl prepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTAbuffer (1 μg total DNA); 100 μl 2.5 M CaCl₂; and, 10 μl 0.1 Mspermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

The sample plates are bombarded at level #4 in a particle gun. Allsamples receive a single shot at 650 PSI, with a total of ten aliquotstaken from each tube of prepared particles/DNA.

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.

Plants are monitored and scored for the appropriate marker, such as thecontrol of Lepidoptera and have insecticidal activity. For example, aFAW feeding assay could be preformed. In such assays, leaf discs fromthe transgenic plant are excised using a 1 cm cork borer or leaf punch.Six leaf discs are prepared for each plant. The leaves are placed in a24 well microtiter plate on top of 500 ul of 0.8% agar. Each leaf discis infested with 2 neonate Fall armyworm and the plate is then sealedwith mylar. A small ventilation hole is made for each well and theplates are then stored in a 28 C growth chamber. The assay is scored formortality, stunting, and leaf consumption at 96 hours.

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added aftersterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCl, 0.10 g/l pyridoxine HCl, and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite (addedafter bringing to volume with D-I H₂O); and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCl, 0.10 g/l pyridoxine HCl, and 0.40 g/lglycine brought to volume with polished D-I H₂O), 0.1 g/l myo-inositol,and 40.0 g/l sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 3 Agrobacterium-Mediated Transformation of Maize

For Agrobacterium-mediated transformation of maize with a silencingelement of the invention, the method of Zhao is employed (U.S. Pat. No.5,981,840, and PCT patent publication WO98/32326; the contents of whichare hereby incorporated by reference). Such as a construct can comprise2 identical 2-300 bp segments of the target gene in oppositeorientations with an “intron” segment between them acting as a hairpinloop. Such a construct can be linked to the dMMB promoter. Briefly,immature embryos are isolated from maize and the embryos contacted witha suspension of Agrobacterium, where the bacteria are capable oftransferring the polynucleotide comprising the silencing element to atleast one cell of at least one of the immature embryos (step 1: theinfection step). In this step the immature embryos are immersed in anAgrobacterium suspension for the initiation of inoculation. The embryosare co-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). The immature embryos are cultured on solid mediumfollowing the infection step. Following this co-cultivation period anoptional “resting” step is contemplated. In this resting step, theembryos are incubated in the presence of at least one antibiotic knownto inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step). Theimmature embryos are cultured on solid medium with antibiotic, butwithout a selecting agent, for elimination of Agrobacterium and for aresting phase for the infected cells. Next, inoculated embryos arecultured on medium containing a selective agent and growing transformedcallus is recovered (step 4: the selection step). The immature embryosare cultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium are cultured on solid medium to regenerate the plants.

Example 4 Soybean Embryo Transformation

Culture Conditions

Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35ml liquid medium SB196 (see recipes below) on rotary shaker, 150 rpm,26° C. with cool white fluorescent lights on 16:8 hr day/nightphotoperiod at light intensity of 60-85 μE/m2/s. Cultures aresubcultured every 7 days to two weeks by inoculating approximately 35 mgof tissue into 35 ml of fresh liquid SB196 (the preferred subcultureinterval is every 7 days).

Soybean embryogenic suspension cultures are transformed with theplasmids and DNA fragments described in the examples above by the methodof particle gun bombardment (Klein et al. (1987) Nature, 327:70).

Soybean Embryogenic Suspension Culture Initiation

Soybean cultures are initiated twice each month with 5-7 days betweeneach initiation.

Pods with immature seeds from available soybean plants 45-55 days afterplanting are picked, removed from their shells and placed into asterilized magenta box. The soybean seeds are sterilized by shaking themfor 15 minutes in a 5% Clorox solution with 1 drop of ivory soap (95 mlof autoclaved distilled water plus 5 ml Clorox and 1 drop of soap). Mixwell. Seeds are rinsed using 2 l-liter bottles of sterile distilledwater and those less than 4 mm are placed on individual microscopeslides. The small end of the seed are cut and the cotyledons pressed outof the seed coat. Cotyledons are transferred to plates containing SB1medium (25-30 cotyledons per plate). Plates are wrapped with fiber tapeand stored for 8 weeks. After this time secondary embryos are cut andplaced into SB 196 liquid media for 7 days.

Preparation of DNA for Bombardment

Either an intact plasmid or a DNA plasmid fragment containing the genesof interest and the selectable marker gene are used for bombardment.Plasmid DNA for bombardment are routinely prepared and purified usingthe method described in the Promega™ Protocols and Applications Guide,Second Edition (page 106). Fragments of the plasmids carrying thesilencing element of interest are obtained by gel isolation of doubledigested plasmids. In each case, 100 ug of plasmid DNA is digested in0.5 ml of the specific enzyme mix that is appropriate for the plasmid ofinterest. The resulting DNA fragments are separated by gelelectrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker MolecularApplications) and the DNA fragments containing silencing element ofinterest are cut from the agarose gel. DNA is purified from the agaroseusing the GELase digesting enzyme following the manufacturer's protocol.

A 50 μl aliquot of sterile distilled water containing 3 mg of goldparticles (3 mg gold) is added to 5 μl of a 1 μg/l DNA solution (eitherintact plasmid or DNA fragment prepared as described above), 50 μl 2.5 MCaCl₂ and 20 μl of 0.1 M spermidine. The mixture is shaken 3 min onlevel 3 of a vortex shaker and spun for 10 sec in a bench microfuge.After a wash with 400 μl 100% ethanol the pellet is suspended bysonication in 40 μl of 100% ethanol. Five μl of DNA suspension isdispensed to each flying disk of the Biolistic PDS1000/HE instrumentdisk. Each 5 μl aliquot contains approximately 0.375 mg gold perbombardment (i.e. per disk).

Tissue Preparation and Bombardment with DNA

Approximately 150-200 mg of 7 day old embryonic suspension cultures areplaced in an empty, sterile 60×15 mm petri dish and the dish coveredwith plastic mesh. Tissue is bombarded 1 or 2 shots per plate withmembrane rupture pressure set at 1100 PSI and the chamber evacuated to avacuum of 27-28 inches of mercury. Tissue is placed approximately 3.5inches from the retaining/stopping screen.

Selection of Transformed Embryos

Transformed embryos were selected either using hygromycin (when thehygromycin phosphotransferase, HPT, gene was used as the selectablemarker) or chlorsulfuron (when the acetolactate synthase, ALS, gene wasused as the selectable marker).

Hygromycin (HPT) Selection

Following bombardment, the tissue is placed into fresh SB196 media andcultured as described above. Six days post-bombardment, the SB196 isexchanged with fresh SB196 containing a selection agent of 30 mg/Lhygromycin. The selection media is refreshed weekly. Four to six weekspost selection, green, transformed tissue may be observed growing fromuntransformed, necrotic embryogenic clusters. Isolated, green tissue isremoved and inoculated into multiwell plates to generate new, clonallypropagated, transformed embryogenic suspension cultures.

Chlorsulfuron (ALS) Selection

Following bombardment, the tissue is divided between 2 flasks with freshSB196 media and cultured as described above. Six to seven dayspost-bombardment, the SB196 is exchanged with fresh SB196 containingselection agent of 100 ng/ml Chlorsulfuron. The selection media isrefreshed weekly. Four to six weeks post selection, green, transformedtissue may be observed growing from untransformed, necrotic embryogenicclusters. Isolated, green tissue is removed and inoculated intomultiwell plates containing SB196 to generate new, clonally propagated,transformed embryogenic suspension cultures.

Regeneration of Soybean Somatic Embryos into Plants

In order to obtain whole plants from embryogenic suspension cultures,the tissue must be regenerated.

Embryo Maturation

Embryos are cultured for 4-6 weeks at 26° C. in SB196 under cool whitefluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro(Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with lightintensity of 90-120 uE/m2s. After this time embryo clusters are removedto a solid agar media, SB166, for 1-2 weeks. Clusters are thensubcultured to medium SB103 for 3 weeks. During this period, individualembryos can be removed from the clusters and screened for theappropriate marker or the ability of the plant, when injected with thesilencing elements, to control the Lepidoptera.

Embryo Desiccation and Germination

Matured individual embryos are desiccated by placing them into an empty,small petri dish (35×10 mm) for approximately 4-7 days. The plates aresealed with fiber tape (creating a small humidity chamber). Desiccatedembryos are planted into SB71-4 medium where they were left to germinateunder the same culture conditions described above. Germinated plantletsare removed from germination medium and rinsed thoroughly with water andthen planted in Redi-Earth in 24-cell pack tray, covered with clearplastic dome. After 2 weeks the dome is removed and plants hardened offfor a further week. If plantlets looked hardy they are transplanted to10″ pot of Redi-Earth with up to 3 plantlets per pot.

Media Recipes SB 196 - FN Lite liquid proliferation medium (per liter) -MS FeEDTA - 100x Stock 1 10 ml MS Sulfate - 100x Stock 2 10 ml FN LiteHalides - 100x Stock 3 10 ml FN Lite P, B, Mo - 100x Stock 4 10 ml B5vitamins (1 ml/L) 1.0 ml 2,4-D (10 mg/L final concentration) 1.0 ml KNO32.83 gm (NH4)2 SO 4 0.463 gm Asparagine 1.0 gm Sucrose (1%) 10 gm pH 5.8FN Lite Stock Solutions Stock # 1000 ml 500 ml 1 MS Fe EDTA 100x StockNa₂ EDTA* 3.724 g  1.862 g  FeSO₄—7H₂O 2.784 g  1.392 g  2 MS Sulfate100x stock MgSO₄—7H₂O 37.0 g 18.5 g MnSO₄—H₂O 1.69 g 0.845 g  ZnSO₄—7H₂O0.86 g 0.43 g CuSO₄—5H₂O 0.0025 g  0.00125 g   3 FN Lite Halides 100xStock CaCl₂—2H₂O 30.0 g 15.0 g KI 0.083 g  0.0715 g  CoCl₂—6H₂O 0.0025g  0.00125 g   4 FN Lite P, B, Mo 100x Stock KH₂PO₄ 18.5 g 9.25 g H₃BO₃0.62 g 0.31 g Na₂MoO₄—2H₂O 0.025 g  0.0125 g  *Add first, dissolve indark bottle while stirring

SB1 solid medium (per liter) comprises: 1 pkg. MS salts(Gibco/BRL—Cat#11117-066); 1 ml B5 vitamins 1000× stock; 31.5 g sucrose;2 ml 2,4-D (20 mg/L final concentration); pH 5.7; and, 8 g TC agar.

SB 166 solid medium (per liter) comprises: 1 pkg. MS salts(Gibco/BRL—Cat#11117-066); 1 ml B5 vitamins 1000× stock; 60 g maltose;750 mg MgCl2 hexahydrate; 5 g activated charcoal; pH 5.7; and, 2 ggelrite.

SB 103 solid medium (per liter) comprises: 1 pkg. MS salts(Gibco/BRL—Cat#11117-066); 1 ml B5 vitamins 1000× stock; 60 g maltose;750 mg MgCl2 hexahydrate; pH 5.7; and, 2 g gelrite.

SB 71-4 solid medium (per liter) comprises: 1 bottle Gamborg's B5 saltsw/sucrose (Gibco/BRL—Cat#21153-036); pH 5.7; and, 5 g TC agar.

2,4-D stock is obtained premade from Phytotech cat# D 295—concentrationis 1 mg/ml.

B5 Vitamins Stock (per 100 ml) which is stored in aliquots at −20 Ccomprises: 10 g myo-inositol; 100 mg nicotinic acid; 100 mg pyridoxineHCl; and, 1 g thiamine. If the solution does not dissolve quicklyenough, apply a low level of heat via the hot stir plate. ChlorsulfuronStock comprises 1 mg/ml in 0.01 N Ammonium Hydroxide

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

The invention claimed is:
 1. A plant cell having stably incorporatedinto its genome a heterologous polynucleotide encoding a double strandedRNA, wherein said double stranded RNA comprises a sense and an antisensestrand, wherein the antisense strand comprises at least 20 consecutivenucleotides fully complementary to the target gene sequence set forth inSEQ ID NO: 6, and wherein the sense strand is fully complementary to theantisense strand, wherein said double stranded RNA, when ingested by apest from the Lepidoptera order, controls the pest from the Lepidopteraorder.
 2. The plant cell of claim 1, wherein said pest comprisesSpodoptera frugiperda.
 3. The plant cell of claim 1, wherein said doublestranded RNA comprises a) the sense and antisense sequence of thesequence set forth in SEQ ID NO: 93, 96, or 99; or b) the sequences setforth in SEQ ID NO: 94, and 95, 97, and 98, or 100 and
 101. 4. The plantcell of claim 1, wherein said double stranded RNA comprises a hairpinRNA.
 5. The plant cell of claim 4, wherein said double stranded RNAcomprises a first segment, a second segment, and a third segment,wherein a) said first segment comprises at least 20 consecutivenucleotides fully complementary to the target sequence set forth in SEQID NO: 6; b) said second segment comprises a loop of sufficient lengthto allow the double stranded RNA to be transcribed as a hairpin RNA;and, c) said third segment comprises at least about 20 consecutivenucleotides fully complementary to the first segment.
 6. The plant cellof claim 1, wherein said polynucleotide is operably linked to aheterologous promoter.
 7. The plant cell of claim 1, wherein said plantcell has stably incorporated into its genome a second polynucleotidecomprising a suppressor enhancer element comprising the target pestsequence or an active variant or fragment thereof, wherein the combinedexpression of the double stranded RNA and the suppressor enhancerelement increases the concentration of an inhibitory RNAi specific forthe pest target sequence in said plant cell.
 8. The plant cell of claim1, wherein said plant cell is from a monocot.
 9. The plant cell of claim8, wherein said monocot is maize, barley, millet, wheat or rice.
 10. Theplant cell of claim 1, wherein said plant cell is from a dicot.
 11. Theplant cell of claim 10, wherein said dicot is soybean, canola, alfalfa,sunflower, safflower, tobacco, Arabidopsis, or cotton.
 12. A plant orplant part comprising a plant cell of claim
 1. 13. The plant or plantpart of claim 7, wherein the combined expression of said double strandedRNA and the suppressor enhancer element increases the concentration ofan inhibitory RNA specific for the pest target sequence in the phloem ofsaid plant or plant part.
 14. A transgenic seed from the plant of claim12.
 15. A method for controlling Lepidoptera comprising feeding to aLepidoptera a composition comprising a double stranded RNA, wherein thedouble stranded RNA comprises a sense and an antisense strand, whereinthe antisense strand comprises at least 20 consecutive nucleotides fullycomplementary to the target gene sequence set forth in SEQ ID NO: 6, andwherein the sense strand is fully complementary to the antisense strand,wherein said double stranded RNA, when ingested by said Lepidopteracontrols the Lepidoptera.
 16. The method of claim 15, wherein saidcomposition comprises a plant or plant part having stably incorporatedinto its genome a polynucleotide encoding said double stranded RNA. 17.The method of claim 15, wherein said pest comprises Spodopterafrugiperda.
 18. The method of claim 15, wherein said double stranded RNAcomprises: a) the sense and antisense sequence of the sequence set forthin SEQ ID NO: 93, 96, or 99; or b) the sequences set forth in SEQ ID NO:94 and 95, 97 and 98, or 100 and
 101. 19. The method of claim 15,wherein said double stranded RNA comprises a hairpin RNA.
 20. The methodof claim 19 wherein said double stranded RNA comprises a first segment,a second segment, and a third segment, wherein a) said first segmentcomprises at least 20 consecutive nucleotides fully complementary to thetarget polynucleotide; b) said second segment comprises a loop ofsufficient length to allow the double stranded RNA to be transcribed asa hairpin RNA; and, c) said third segment comprises at least 20consecutive nucleotides fully complementary to the first segment. 21.The method of claim 16, wherein said plant or plant part has stablyincorporated into its genome a second polynucleotide comprising asuppressor enhancer element comprising the target pest sequence or anactive variant or fragment thereof, wherein the combined expression ofthe double stranded RNA and the suppressor enhancer element increasesthe concentration of an inhibitory RNAi specific for the pest targetsequence in said plant cell.
 22. The method of claim 15, wherein saidplant is a monocot.
 23. The method of claim 22, wherein said monocot ismaize, barley, millet, wheat or rice.
 24. The method of claim 15,wherein said plant is a dicot.
 25. The method of claim 24, wherein saidplant is soybean, canola, alfalfa, sunflower, safflower, tobacco,Arabidopsis, or cotton.